Antibodies that bind OV064 and methods of use therefor

ABSTRACT

Antibodies and antigen-binding fragments of antibodies that bind OV064 are disclosed. The antibodies bind an extracellular domain of OV064. Some of the antibodies and antigen-binding fragments bind an epitope on OV064 sufficient to induce internalization. In some embodiments, the antibodies are humanized, chimeric or human. Nucleic acids and vectors encoding the antibodies or portions thereof, recombinant cells that contain the nucleic acids, and compositions comprising the antibodies or antigen-binding fragments are also disclosed. The invention also provides therapeutic and diagnostic methods utilizing the antibodies and antigen-binding fragments provided herein.

RELATED APPLICATIONS

The present application is a Divisional of U.S. application Ser. No.11/886,434 (published), filed Feb. 20, 2009, which is a National Stageof International Application Serial No. PCT/US06/08892, filed Mar. 10,2006, and claims priority to U.S. Provisional Application Ser. No.60/664,828 (closed), filed Mar. 24, 2005, the entire contents of whichare incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to antibodies and antigen bindingfragments thereof which bind OV064, as well as therapeutic anddiagnostic methods of use and compositions comprising the describedantibodies and antigen binding fragments.

BACKGROUND OF THE INVENTION

Despite advances in the field of cancer therapy, tumor cell resistanceto chemotherapeutic agents remains a significant problem in clinicaloncology, as one of the main reasons many prevalent forms of humancancer resist effective chemotherapeutic intervention. Cell surfaceproteins expressed on cancer cells are presently being utilized fortargeting specific cancer therapy in several tumor types. For example,Panorex® (edrocolomab), a monoclonal antibody which targets metastaticcolon cancer, was the first monoclonal antibody approved for cancertreatment. Additional monoclonal antibody therapies have been approvedin other cancers, for example, Herceptin® (trastuzumab), which is anantibody against the Her2/neu antigen, is currently being used to treatbreast cancer (Finn and Slamon, Cancer Chemother Biol Response Modif 21:223-233 (2003)). RituxanMabThera® (rituximab), which binds to the CD20antigen, is being used to treat B-cell lymphomas (Multani and White,Cancer Chemother Biol Response Modif. 21: 235-258 (2003)). Furthermore,several monoclonal antibody candidates presently undergoing clinicaltrials are showing great promise in colon and prostate cancers (Galskyet al., Phase I trial of MLN2704 in patients with castrate-metastaticprostate cancer (CMPC). ASCO Annual Meeting (2004); Hoff et al.,Oncology (Huntingt) 18: 736-741; discussion 742, 745-736 (2004);Milowsky et al., J Clin Oncol 22: 2522-2531 (2004)). Many antibodiesthat are currently in development for cancer indications areunconjugated human or humanized IgG molecules that illicit the desiredeffect by inhibiting growth factor mediated proliferation or induceantibody dependent cell mediated cytotoxicity (ADCC) on cells expressingthe targeted antigen. Any individual target of interest may not beappropriate for use in antibody targeted therapeutics or applicable foreffective therapies in all cancers, however.

Additional approaches to antibody targeted therapeutics includeutilization of tumor specific expression of a cell surface antigen totarget toxins to the tumor site using monoclonal antibody-drugimmunoconjugates (Payne, Cancer Cell 3: 207-212 (2003)). However, incommon with chemotherapeutic approaches, immunotoxin therapy alsosuffers from significant drawbacks when applied to solid tumors. Forexample, antigen-negative or antigen-deficient cells can survive andrepopulate the tumor or lead to further metastases. An additional reasonfor solid tumor resistance to antibody-based therapies is that the tumormass is generally impermeable to macromolecular agents such asantibodies and immunotoxins (Burrows et al., Cancer Res. 52: 5954-62(1992); Dvorak et al., Cancer Cells. 3: 77-85 (1991); Baxter and Jain,Microvasc Res. 41: 5-23 (1991)). Both the physical diffusion distancesand the interstitial pressure within the tumor are significantlimitations to this type of therapy. Therefore, solid tumors, which makeup over 90% of all human cancers, have so far proven resistant toimmunotoxin and antibody treatment. Further development and efficacy inclinical trials with useful antigens will be valuable advancements inidentifying additional effective cancer therapies. Particularly usefulare target antigens that have the ability to internalize and release thetoxin inside the tumor cell and induce cell killing (Payne, Cancer Cell3: 207-212 (2003)). Additionally, particularly useful targets would beprimarily expressed in tumors, while lacking non-specific tissueexpression. This approach has been shown to work very well inpreclinical models and in vitro on cells expressing the antigen. Todate, a couple of antibodies are in clinical development that areconjugated with the microtubule inhibitor maytansinoid, or DM1 (Tassoneet al., Cancer Res 64: 4629-4636 (2004); Tolcher et al., J Clin Oncol21: 211-222 (2003).

In view of the shortcoming of existing cancer therapies, there exists aneed for identification of additional and improved modalities forameliorating and treating cancers. The identification of additionaleffective targets is thus useful in conjunction with development ofantibody targeted therapeutics and is certain to aid in theidentification and generation of novel therapeutic regimens for cancertreatment.

DESCRIPTION OF THE FIGURE

FIG. 1 depicts the structure of the complete combination heavy and lightchain immunoglobulin DNA cassette antibody expression vector pLKTOK58D.

DESCRIPTION OF THE INVENTION

The present invention relates to methods of using compositionscomprising antibodies or antigen binding fragments which target OV064for the treatment of cancers. In particular, the provided methodscomprise treating a subject having a cancer by administering to asubject a sufficient amount of an immunoconjugate to treat the subject'scancer. The methods comprise utilizing an immunoconjugate whichcomprises an antibody or an antigen binding fragment and a cytotoxicmoiety. The antibody or antigen binding fragment of the immunoconjugatebinds specifically to an epitope on OV064 selected from any one of: anepitope comprising amino acid residues selected from amino acid residues167-176 of OV064, an epitope comprising amino acid residues selectedfrom amino acid residues 177-181 of OV064, an epitope comprising aminoacid residues selected from amino acid residues 238-257 of OV064, anepitope recognized by sc77, an epitope recognized by sc 189, an epitoperecognized by sc209, an epitope recognized by 4G10, an epitoperecognized by 3A4, or an epitope recognized by 2F3. Binding of theimmunoconjugate to a cell expressing OV064 induces internalization ofthe immunoconjugate and OV064 into the cell. The cytotoxic moiety of theimmunoconjugate can comprise a radioisotope, a therapeutic agent, or atumor-activated prodrug. In some apects, the cytotoxic moiety is atherapeutic agent comprising a maytansine, an auristatin, a dolastatin,a duocarmycin, a cryptophycin, a taxane, a DNA alkylating agent,calicheamicin, or a derivative of any one of these.

The invention also relates to antibodies and antigen-binding fragmentsof antibodies which bind specifically to the extracellular domain ofOV064 sufficient to induce internalization of the antibody or theantigen binding fragment thereof and OV064 into the cell. The antibodyor the antigen binding fragments of the invention bind specifically toan epitope on OV064 selected from any one of: an epitope comprisingamino acid residues selected from amino acid residues 167-176 of OV064,an epitope comprising amino acid residues selected from amino acidresidues 177-181 of OV064, an epitope comprising amino acid residuesselected from amino acid residues 238-257 of OV064, an epitoperecognized by sc77, an epitope recognized by sc189, an epitoperecognized by sc209, an epitope recognized by 4G10, an epitoperecognized by 3A4, or an epitope recognized by 2F3. Further provided areantibodies or antigen binding fragments which bind specifically to anepitope comprising amino acid residues 67-76 of OV064, or an epitoperecognized by 8G5. Human and modified antibodies and antigen bindingfragments thereof are provided. For example, humanized and chimericantibodies, and antigen binding fragments of the foregoing areencompassed in the invention.

The invention provides immunoconjugates comprising an antibody orantigen-binding fragment thereof coupled to a cytotoxic moiety that hasthe ability to kill cells expressing OV064. The antibody or the antigenbinding fragment of the immunoconjugates bind specifically to theextracellular domain of OV064 sufficient to induce internalization ofthe antibody or the antigen binding fragment thereof and OV064 into thecell. The antibody or the antigen binding fragments of theimmunoconjugates bind specifically to an epitope on OV064 selected fromany one of: an epitope comprising amino acid residues selected fromamino acid residues 167-176 of OV064, an epitope comprising amino acidresidues selected from amino acid residues 177-181 of OV064, an epitopecomprising amino acid residues selected from amino acid residues 238-257of OV064, an epitope recognized by sc77, an epitope recognized by sc189,an epitope recognized by sc209, an epitope recognized by 4G10, anepitope recognized by 3A4, or an epitope recognized by 2F3. The providedimmunoconjugates may comprise any one of the antibodies or antigenbinding fragments of the invention. The cytotoxic moiety of theimmunoconjugates can comprise a radioisotope, a therapeutic agent, or atumor-activated prodrug. In one aspect, the immunoconjugate comprisingan antibody or antigen-binding fragment thereof of the invention canreduce tumor-cell induced immune suppression of tumor cells expressingOV064. In an additional aspect, the immunoconjugate comprising anantibody or antigen-binding fragment thereof can inhibit tumor growth.In some aspects, the immunoconjugate comprises an antibody or an antigenbinding fragment of the invention conjugated to a matansine, anauristatin, a dolastatin, a duocarmycin, a cryptophicin, a taxol, a DNAalkylating agent, or a calicheamicin or a derivative of any of theforegoing. In certain aspects, maytasines of the immunoconjugates of theinvention comprise DM1 or DM4 conjugated to an antibody or an antigenbinding fragment of the invention.

In still additional aspects, the antibody or antigen-binding fragmentcan inhibit OV064-mediated cell signaling of a first cell expressing anOV064 by inhibiting interaction with a second cell bearing a ligand ofOV064, such as T-cells □. In some aspects, the antibody orantigen-binding fragment competitively inhibits binding of one or moreof the antibodies provided and described herein (e.g., mAb 4G10, mAb3A4, mAb 2F3, mAb sc77, mAb sc189, and/or mAb sc209).

In particular, one embodiment of the invention provides a method oftreating cancer in a subject. The method comprises administering to asubject an immunoconjugate in an effective amount so as to treat thecancer. The methods comprise utilizing an immunoconjugate whichcomprises an antibody or an antigen binding fragment and a cytotoxicmoiety. The antibody or the antigen binding fragment of theimmunoconjugate binds specifically to an epitope on OV064 selected fromany one of: an epitope comprising amino acid residues selected fromamino acid residues 167-176 of OV064, an epitope comprising amino acidresidues selected from amino acid residues 177-181 of OV064, an epitopecomprising amino acid residues selected from amino acid residues 238-257of OV064, an epitope recognized by sc77, an epitope recognized by sc189,an epitope recognized by sc209, an epitope recognized by 4G10, anepitope recognized by 3A4, or an epitope recognized by 2F3. Binding ofthe immunoconjugate to a cell expressing OV064 induces internalizationof the immunoconjugate and OV064 into the cell, and administration ofthe effective amount of immunoconjugate, is sufficient to reduce orinhibit the growth of the subject's tumor.

In certain embodiments, the cytotoxic moiety of the immunoconjugatecomprises a radioisotope, a therapeutic agent, or a tumor activatedpro-drug. Where the cytotoxic moiety is a therapeutic agent, the agentcan comprise a maytansine, an auristatin, a duocarmycin, a dolastatin, acryptophycin, a taxol, a DNA alkylating agent, or calicheamicin, or aderivative of any of the foregoing. In some aspects the therapeuticagent is a maytansine which is DM1 or DM4.

The invention also relates to methods of inhibiting OV064 mediated cellsignaling resulting from interaction of a cell expressing OV064 with anOV064 receptor. For example, the method comprises contacting a cell witha sufficient amount of an antibody or an antigen-binding fragment of theinvention which binds specifically to an epitope on OV064 selected fromany one of: an epitope comprising amino acid residues selected fromamino acid residues 167-176 of OV064, an epitope comprising amino acidresidues selected from amino acid residues 177-181 of OV064, an epitopecomprising amino acid residues selected from amino acid residues 238-257of OV064, an epitope recognized by sc77, an epitope recognized by sc189,an epitope recognized by sc209, an epitope recognized by 4G10, anepitope recognized by 3A4, or an epitope recognized by 2F3. Binding ofthe antibody or antigen binding fragment thereof to a cell expressingOV064 induces internalization of the antibody and OV064 into the cell,and reduces the amount of OV064 available for interaction with the OV064receptor. The method thereby results in inhibiting OV064 mediated cellsignaling resulting from interaction of a cell expressing OV064 with anOV064 receptor. Provided methods of the invention also include a methodof killing a cell expressing OV064. For example, an antibody or anantigen binding fragment of the invention which is an immunoconjugatecomprising a cytotoxic moiety may be used in the method. Alternatively,an antibody or an antigen binding fragment which is bound to a secondaryantibody or antigen binding fragment which comprises an immunoconjugatecoupled to a cytotoxic moiety may be used in the method. For example,the method comprises contacting a cell expressing OV064 with aneffective amount of an antibody or an antigen binding fragment of theinvention which binds the extracellular domain of OV064, and which is animmunoconjugate comprising a cytotoxic moiety or is bound to a secondaryantibody which is an immunoconjugate comprising a cytotoxic moiety.Binding of the antibody or antigen binding fragment to the cell inducesinternalization of the antibody or antigen binding fragment and OV064into the cell, and internalization is sufficient to deliver toxicpayload into the cell, thereby killing the cell. The methods of eitherinhibition of OV064 cell signaling, or killing a OV064 expressing cellare useful in cells expressing OV064. For example, cells include ES-2ovarian carcinoma cells, SKBR-3 breast carcinoma cells, ZR75-1 cells,OVCAR3 cells, MDA-MB-468 cells, MCF-7 cells, DLD1 cells, fresh or frozenovarian tumor cells, or cells comprising a recombinant nucleic acidencoding OV064 or a portion thereof. In certain aspects the OV064receptor is BTLA. The method of inhibition of signaling, or the methodof killing cells expressing OV064 may be carries out in vitro, in vivo,ex vivo, or in situ.

The methods comprise use of an antibody or antigen binding fragmentwhich binds the extracellular domain of OV064, as well as use ofimmunoconjugates comprising an antibody or an antigen binding fragmentthereof which binds the extracellular domain of OV064. Thus, providedare antibodies or antigen binding fragments thereof for use in thecompositions (e.g., immunoconjugates) and in the methods providedherein. The invention encompasses the provided antibodies or antigenbinding fragments thereof, immunoconjugates of the provided antibodiesor antigen binding fragments thereof, as well as uses of any of theprovided antibodies or antigen binding fragments, or immunoconjugatescomprising the antibodies or the antigen binding fragments inconjunction with the described methods. Use of the provided antibodiesor antigen binding fragments or any combination of the providedantibodies or antigen binding fragments in compositions and/or methodsof the invention provided is intended. Provided antibodies or antigenbinding fragments include an antibody or antigen binding fragmentthereof which binds specifically to an epitope on OV064 selected fromany one of: an epitope comprising amino acid residues selected fromamino acid residues 167-176 of OV064, an epitope comprising amino acidresidues selected from amino acid residues 177-181 of OV064, an epitopecomprising amino acid residues selected from amino acid residues 238-257of OV064, an epitope recognized by sc77, an epitope recognized by sc189,an epitope recognized by sc209, an epitope recognized by 4G10, anepitope recognized by 3A4, or an epitope recognized by 2F3. Binding ofthe antibody or the antigen binding fragment to a cell expressing OV064induces internalization of the antibody or the antigen binding fragmentand OV064 into the cell. Also included in the invention is an antibodyor antigen binding fragment which binds specifically to an epitope onOV064 which is an epitope recognized by 8G5, or an epitope comprisingamino acid residues selected from amino acid residues 67-71 of OV064.

Provided antibody or antigen binding fragments compete with one or moreof sc77, sc189, sc209, 4G10, 3A4, or 2F3 for binding to OV064.Additional human antibody or antigen binding fragments compete with 8G5for binding to OV064.

In certain embodiments, the antibody or antigen binding fragment thereofcomprises the ability to kill cells expressing OV064 when coupled to acytotoxic moiety. In other embodiments, the antibody or antigen-bindingfragment comprises the ability to reduce tumor-cell induced immunesuppression of tumor cells expressing OV064.

In some embodiments the antibody or the antigen binding fragment is ahuman antibody or antigen binding fragment thereof.

In additional embodiments, the antibody may be a modified antibody or anantigen binding fragment thereof. Where the antibody or the antigenbinding fragment is modified, it may be a modified rodent, rabbit, orhuman antibody or antigen binding fragment. In one aspect, the rodentantibody or antigen binding fragment is a murine antibody or antigenbinding fragment. In certain aspects, the modified antibody or antigenbinding fragment comprises a CDR-grafted antibody, a humanized antibody,or a chimeric antibody, or an antigen binding fragment or fragments ofthe foregoing. Certain aspects include a humanized or chimeric antibodyor an antigen-binding fragment thereof having binding specificity for anepitope on OV064 selected from any one of: an epitope comprising aminoacid residues selected from amino acid residues 167-176 of OV064, anepitope comprising amino acid residues selected from amino acid residues177-181 of OV064, an epitope comprising amino acid residues selectedfrom amino acid residues 238-257 of OV064, an epitope recognized bysc77, an epitope recognized by sc189, an epitope recognized by sc209, anepitope recognized by 4G10, an epitope recognized by 3A4, or an epitoperecognized by 2F3. In some embodiments, the antibody or antigen bindingfragment thereof comprises a light chain, wherein the light chaincomprises one, two, or three complementarity determining region derivedfrom monoclonal antibody 4G10, 3A4, or 2F3. In additional aspects thelight chain comprises one, two, or three complementarity determiningregion derived from monoclonal antibody 4G10, 3A4, or 2F3 and theframework region sequence is derived from a light chain of human origin.In other embodiments, the heavy chain comprises one, two, or threecomplementarity determining region derived from monoclonal antibody4G10, 3A4, or 2F3. In additional aspects, the heavy chain comprises one,two, or three complementarity determining region derived from monoclonalantibody 4G10, 3A4, or 2F3 and the framework region derived from a heavychain of human origin. In some aspects, the humanized antibody competeswith murine antibody 4G10, 3A4, or 2F3 for binding to OV064. In someaspects the light chain comprises at least one complementaritydetermining region derived from monoclonal antibody 4G10, 3A4, or 2F3and the heavy chain comprises at least one complementarity determiningregion derived from monoclonal antibody 4G10, 3A4, or 2F3. In additionalaspects, the humanized antibody or antigen-binding fragment comprisesall three complementarity determining regions derived from the lightchain of monoclonal antibody 4G10, 3A4, or 2F3; and the heavy chaincomprises all three complementarity determining regions derived from theheavy chain of monoclonal antibody 4G10, 3A4, or 2F3.

In still other embodiments, a humanized antibody competes with murineantibody 8G5 for binding to OV064. In additional aspects the humanizedantibody or the antigen binding fragment comprises one, two or threecomplementarity determining regions derived from the heavy chain ofmonoclonal antibody 8G5 and one, two, or three complementaritydetermining regions derived from the light chain of monoclonal antibody8G5. In some aspects the light chain comprises at least onecomplementarity determining region derived from monoclonal antibody 8G5and the heavy chain comprises at least one complementarity determiningregion derived from monoclonal antibody 8G5. In additional aspects, thehumanized antibody or antigen-binding fragment comprises all threecomplementarity determining regions derived from the light chain ofmonoclonal antibody 8G5; and the heavy chain comprises all threecomplementarity determining regions derived from the heavy chain ofmonoclonal antibody 8G5

In certain embodiments, an antibody or modified antibody has one or morecomplementarity determining regions from the human monoclonal antibodysc77, sc189, or sc209. In additional embodiments, the antibody orantigen binding fragment thereof competes for binding with the sameepitope as monoclonal antibody sc77, sc 189, or sc209, and/or competesfor binding the same epitope as murine monoclonal antibody 4G10, 3A4, or2F3.

For example, in specific embodiments the antibody or antigen-bindingfragment comprises one, two or three heavy chain complementaritydetermining region (HCDR1, HCDR2 and/or HCDR3) of the monoclonalantibody sc77. The complementarity determining region may comprise anamino acid sequence of: HCDR1 (e.g., SEQ ID NO: 13; SEQ ID NO: 13wherein optionally one or two amino acids are conservativelysubstituted); HCDR2 (e.g., SEQ ID NO: 14; SEQ ID NO: 14 whereinoptionally one or two amino acids are conservatively substituted); orHCDR3 (e.g., SEQ ID NO: 15 or SEQ ID NO: 15 wherein optionally one ortwo amino acids are conservatively substituted). In certain aspects, theantibody or antigen-binding fragment comprises all three heavy chaincomplementarity determining regions (HCDR1 (e.g., SEQ ID NO: 13; SEQ IDNO: 13 wherein optionally one or two amino acids are conservativelysubstituted); HCDR2 (e.g., SEQ ID NO: 14; SEQ ID NO: 14 whereinoptionally one or two amino acids are conservatively substituted); andHCDR3 (e.g., SEQ ID NO: 15 or SEQ ID NO: 15 wherein optionally one ortwo amino acids are conservatively substituted)) of the monoclonalantibody sc77. In some embodiments, the heavy chain sequence compriseshuman heavy chain constant region sequence of human isotype IgG1 orIgG2.

In other embodiments, the antibody or the antigen binding fragment maycomprise one, two or three light chain complementarity determiningregion (LCDR1, LCDR2 and/or LCDR3) of the monoclonal antibody sc77. Thecomplementarity determining region may comprise an amino acid sequenceof: LCDR1 (e.g., SEQ ID NO: 22, SEQ ID NO: 22 wherein optionally one ortwo amino acids are conservatively substituted); LCDR2 (e.g., SEQ ID NO:23, SEQ ID NO: 23 wherein one or two amino acids are conservativelysubstituted); or LCDR3 (e.g., SEQ ID NO: 24, SEQ ID NO: 24 whereinoptionally one or two amino acids are conservatively substituted). Incertain aspects, the antibody or the antigen binding fragment comprisesall three light chain complementarity determining regions (LCDR1 (e.g.,SEQ ID NO: 22, SEQ ID NO: 22 wherein optionally one or two amino acidsare conservatively substituted); LCDR2 (e.g., SEQ ID NO: 23, SEQ ID NO:23 wherein one or two amino acids are conservatively substituted); andLCDR3 (e.g., SEQ ID NO: 24, SEQ ID NO: 24 wherein optionally one or twoamino acids are conservatively substituted)) of the monoclonal antibodysc77.

In certain aspects, the antibody or the antigen binding fragmentcomprises one, two or three heavy chain complementarity determiningregion (HCDR1 (e.g., SEQ ID NO: 13; SEQ ID NO: 13 wherein optionally oneor two amino acids are conservatively substituted); HCDR2 (e.g., SEQ IDNO: 14; SEQ ID NO: 14 wherein optionally one or two amino acids areconservatively substituted); and/or HCDR3 (e.g., SEQ ID NO: 15 or SEQ IDNO: 15 wherein optionally one or two amino acids are conservativelysubstituted)) of the monoclonal antibody sc77; and one, two or threelight chain complementarity determining region (LCDR1 (e.g., SEQ ID NO:22, SEQ ID NO: 22 wherein optionally one or two amino acids areconservatively substituted); LCDR2 (e.g., SEQ ID NO: 23, SEQ ID NO: 23wherein one or two amino acids are conservatively substituted); or LCDR3(e.g., SEQ ID NO: 24, SEQ ID NO: 24 wherein optionally one or two aminoacids are conservatively substituted)) of the monoclonal antibody sc77.In additional aspects, the antibody or the antigen binding fragmentcomprises all three heavy chain complementarity determining regions(HCDR1 (e.g., SEQ ID NO: 13; SEQ ID NO: 13 wherein optionally one or twoamino acids are conservatively substituted); HCDR2 (e.g., SEQ ID NO: 14;SEQ ID NO: 14 wherein optionally one or two amino acids areconservatively substituted); and HCDR3 (e.g., SEQ ID NO: 15 or SEQ IDNO: 15 wherein optionally one or two amino acids are conservativelysubstituted)) of the monoclonal antibody sc77; and all three light chaincomplementarity determining regions (LCDR1 (e.g., SEQ ID NO: 22, SEQ IDNO: 22 wherein optionally one or two amino acids are conservativelysubstituted); LCDR2 (e.g., SEQ ID NO: 23, SEQ ID NO: 23 wherein one ortwo amino acids are conservatively substituted); or LCDR3 (e.g., SEQ IDNO: 24, SEQ ID NO: 24 wherein optionally one or two amino acids areconservatively substituted)) of the monoclonal antibody sc77. In someembodiments, the heavy chain sequence further comprises human heavychain constant region sequence of human isotype IgG1 or IgG2.

In additional aspects, the antibody or the antigen binding fragmentcomprises an amino acid sequence shown as SEQ ID NO:4, an amino acidsequence of the light chain variable region amino acid sequence of theantibody encoded by the purified DNA having ATCC Accession NumberPTA-6294, or the amino acid encoded by the nucleotide sequence shown asnucleic acid residues 15-737 of SEQ ID NO:3.

In other additional aspects, the antibody or the antigen bindingfragment comprises an amino acid sequence shown as SEQ ID NO:2, an aminoacid sequence of the heavy chain variable region amino acid sequence ofthe antibody encoded by the purified DNA having ATCC Accession NumberPTA-6294, or the amino acid sequence encoded by the nucleotide sequenceshown as nucleic acid residues 13-1416 of SEQ ID NO:1.

In still other aspects, the antibody or the antigen binding fragmentcomprises a light chain variable region comprising the amino acidsequence shown as SEQ ID NO:4, or the light chain variable region aminoacid sequence of the antibody encoded by the purified DNA having ATCCAccession Number PTA-6294; and a heavy chain variable region comprisingthe amino acid sequence shown as SEQ ID NO:2, or the heavy chainvariable region amino acid sequence of the antibody encoded by thepurified DNA having ATCC Accession Number PTA-6294. In further aspects,the antibody or antigen binding fragment comprises two heavy and twolight chains.

In some embodiments, the human antibody or antigen binding fragment issc77, or a fragment thereof.

In another example, the antibody or antigen-binding fragment comprisesone, two, or three heavy chain complementarity determining region(HCDR1, HCDR2 and/or HCDR3) of the monoclonal antibody sc 189. Thecomplementarity determining region may comprise an amino acid sequenceof HCDR1 (e.g., SEQ ID NO: 16, SEQ ID NO: 16 wherein optionally one ortwo amino acids are conservatively substituted); HCDR2 (e.g., SEQ ID NO:17, SEQ ID NO: 17 wherein optionally one or two amino acids areconservatively substituted); or HCDR3 (e.g., SEQ ID NO: 18, SEQ ID NO:18 wherein optionally one or two amino acids are conservativelysubstituted). In certain aspects, the antibody or antigen-bindingfragment comprises all three heavy chain complementarity determiningregions (HCDR1 (e.g., SEQ ID NO: 16, SEQ ID NO: 16 wherein optionallyone or two amino acids are conservatively substituted); HCDR2 (e.g., SEQID NO: 17, SEQ ID NO: 17 wherein optionally one or two amino acids areconservatively substituted); and HCDR3 (e.g., SEQ ID NO: 18, SEQ ID NO:18 wherein optionally one or two amino acids are conservativelysubstituted)) of the monoclonal antibody sc189. In some embodiments, theheavy chain sequence further comprises human heavy chain constant regionsequence of human isotype IgG1 or IgG2.

In other embodiments, the antibody or antigen binding fragment maycomprise one, two, or three light chain complementarity determiningregion (LCDR1, LCDR2 and/or LCDR3) of the monoclonal antibody sc189. Thecomplementarity determining region may comprise an amino acid sequenceof: LCDR1 (e.g., SEQ ID NO: 25, SEQ ID NO: 25 wherein optionally one ortwo amino acids are conservatively substituted); LCDR2 (e.g., SEQ ID NO:26, SEQ ID NO: 26 wherein optionally one or two amino acids areconservatively substituted); or LCDR3 (e.g., SEQ ID NO: 27, SEQ ID NO:27 wherein optionally one or two amino acids are conservativelysubstituted). In certain aspects, the antibody or antigen bindingfragment may comprise all three light chain complementarity determiningregions (LCDR1 (e.g., SEQ ID NO: 25, SEQ ID NO: 25 wherein optionallyone or two amino acids are conservatively substituted); LCDR2 (e.g., SEQID NO: 26, SEQ ID NO: 26 wherein optionally one or two amino acids areconservatively substituted); and LCDR3 (e.g., SEQ ID NO: 27, SEQ ID NO:27 wherein optionally one or two amino acids are conservativelysubstituted)) of the monoclonal antibody sc189.

In certain aspects, the antibody or antigen binding fragment comprisesone, two, or three heavy chain complementarity determining region (HCDR1(e.g., SEQ ID NO: 16, SEQ ID NO: 16 wherein optionally one or two aminoacids are conservatively substituted); HCDR2 (e.g., SEQ ID NO: 17, SEQID NO: 17 wherein optionally one or two amino acids are conservativelysubstituted); and/or HCDR3 (e.g., SEQ ID NO: 18, SEQ ID NO: 18 whereinoptionally one or two amino acids are conservatively substituted)) ofthe monoclonal antibody sc189; and one, two, or three light chaincomplementarity determining region (LCDR1 (e.g., SEQ ID NO: 25, SEQ IDNO: 25 wherein optionally one or two amino acids are conservativelysubstituted); LCDR2 (e.g., SEQ ID NO: 26, SEQ ID NO: 26 whereinoptionally one or two amino acids are conservatively substituted);and/or LCDR3 (e.g., SEQ ID NO: 27, SEQ ID NO: 27 wherein optionally oneor two amino acids are conservatively substituted)) of the monoclonalantibody sc189. In additional aspects, the antibody or antigen bindingfragment comprises all three heavy chain complementarity determiningregions (HCDR1 (e.g., SEQ ID NO: 16, SEQ ID NO: 16 wherein optionallyone or two amino acids are conservatively substituted); HCDR2 (e.g., SEQID NO: 17, SEQ ID NO: 17 wherein optionally one or two amino acids areconservatively substituted); and HCDR3 (e.g., SEQ ID NO: 18, SEQ ID NO:18 wherein optionally one or two amino acids are conservativelysubstituted)) of the monoclonal antibody sc189; and all three lightchain complementarity determining regions (LCDR1 (e.g., SEQ ID NO: 25,SEQ ID NO: 25 wherein optionally one or two amino acids areconservatively substituted); LCDR2 (e.g., SEQ ID NO: 26, SEQ ID NO: 26wherein optionally one or two amino acids are conservativelysubstituted); and LCDR3 (e.g., SEQ ID NO: 27, SEQ ID NO: 27 whereinoptionally one or two amino acids are conservatively substituted)) ofthe monoclonal antibody sc 189. In some embodiments, the heavy chainsequence further comprises human heavy chain constant region sequence ofhuman isotype IgG1 or IgG2.

In additional aspects, the antibody or antigen binding fragmentcomprises and amino acid sequence shown as SEQ ID NO:8, an amino acidsequence of the light chain variable region amino acid sequence of theantibody encoded by the purified DNA having ATCC Accession NumberPTA-6295, or the amino encoded by the nucleotide sequence shown asnucleic acid residues 15-734 of SEQ ID NO:7.

In other additional aspects, the antibody or antigen binding fragmentthereof comprises an amino acid sequence shown as SEQ ID NO:6, an aminoacid sequence of the heavy chain variable region amino acid sequence ofthe antibody encoded by the purified DNA having ATCC Accession NumberPTA-6295, or the amino acid sequence encoded by the nucleotide sequenceshown as nucleic acid residues 13-1413 of SEQ ID NO:5.

In still other aspects, the antibody or antigen binding fragmentcomprises a light chain variable region comprising the amino acidsequence shown as SEQ ID NO:8, or the light chain variable region aminoacid sequence of the antibody encoded by the purified DNA having ATCCAccession Number PTA-6295; and a heavy chain variable region comprisingthe amino acid sequence shown as SEQ ID NO:6, or the heavy chainvariable region amino acid sequence of the antibody encoded by thepurified DNA having ATCC Accession Number PTA-6295. In certain aspectsantibody or antigen binding fragment comprises two heavy and two lightchains.

In other embodiments, the human antibody or antigen binding fragment issc189, or a fragment thereof.

Still further provided are specific embodiments wherein the antibody orantigen-binding fragment comprises one, two, or three heavy chaincomplementarity determining region (HCDR1, HCDR2 and/or HCDR3) of themonoclonal antibody sc209. The complementarity determining region maycomprise an amino acid sequence of: HCDR1 (e.g., SEQ ID NO: 19, SEQ IDNO: 19 wherein optionally one or two amino acids are conservativelysubstituted); HCDR2 (e.g., SEQ ID NO: 20, SEQ ID NO: 20 whereinoptionally one or two amino acids are conservatively substituted); orHCDR3 (e.g., SEQ ID NO: 21, SEQ ID NO: 21 wherein optionally one or twoamino acids are conservatively substituted). In certain aspects, theantibody or antigen-binding fragment comprises all three heavy chaincomplementarity determining regions (HCDR1 (e.g., SEQ ID NO: 19, SEQ IDNO: 19 wherein optionally one or two amino acids are conservativelysubstituted); HCDR2 (e.g., SEQ ID NO: 20, SEQ ID NO: 20 whereinoptionally one or two amino acids are conservatively substituted); andHCDR3 (e.g., SEQ ID NO: 21, SEQ ID NO: 21 wherein optionally one or twoamino acids are conservatively substituted)) of the monoclonal antibodysc209. In some embodiments, the heavy chain sequence comprises humanheavy chain constant region sequence of human isotype IgG1 or IgG2.

In other embodiments, the antibody or antigen binding fragment maycomprise one, two or three light chain complementarity determiningregion (LCDR1, LCDR2 and/or LCDR3) of the monoclonal antibody sc209. Thecomplementarity determining region may comprise an amino acid sequenceof: LCDR1, (e.g., SEQ ID NO: 28 or SEQ ID NO: 28 wherein optionally oneor two amino acids are conservatively substituted); LCDR2, (e.g., SEQ IDNO: 29 or SEQ ID NO: 29 wherein optionally one or two amino acids areconservatively substituted); or LCDR3, (e.g., SEQ ID NO: 30 or SEQ IDNO: 30 wherein optionally one or two amino acids are conservativelysubstituted). In certain aspects, the antibody or antigen bindingfragment may comprise all three light chain complementarity determiningregions (LCDR1, (e.g., SEQ ID NO: 28 or SEQ ID NO: 28 wherein optionallyone or two amino acids are conservatively substituted); LCDR2, (e.g.,SEQ ID NO: 29 or SEQ ID NO: 29 wherein optionally one or two amino acidsare conservatively substituted); and LCDR3, (e.g., SEQ ID NO: 30 or SEQID NO: 30 wherein optionally one or two amino acids are conservativelysubstituted)) of the monoclonal antibody sc209.

In certain aspects, the antibody or antigen binding fragment comprisesone, two or three heavy chain complementarity determining region (HCDR1(e.g., SEQ ID NO: 19, SEQ ID NO: 19 wherein optionally one or two aminoacids are conservatively substituted); HCDR2 (e.g., SEQ ID NO: 20, SEQID NO: 20 wherein optionally one or two amino acids are conservativelysubstituted); and/or HCDR3 (e.g., SEQ ID NO: 21, SEQ ID NO: 21 whereinoptionally one or two amino acids are conservatively substituted)) ofthe monoclonal antibody sc209; and one, two, or three chaincomplementarity determining region (LCDR1, (e.g., SEQ ID NO: 28 or SEQID NO: 28 wherein optionally one or two amino acids are conservativelysubstituted); LCDR2, (e.g., SEQ ID NO: 29 or SEQ ID NO: 29 whereinoptionally one or two amino acids are conservatively substituted);and/or LCDR3, (e.g., SEQ ID NO: 30 or SEQ ID NO: 30 wherein optionallyone or two amino acids are conservatively substituted)) of themonoclonal antibody sc209. In additional aspects, the antibody orantigen binding fragment comprises all three heavy chain complementaritydetermining regions (HCDR1 (e.g., SEQ ID NO: 19, SEQ ID NO: 19 whereinoptionally one or two amino acids are conservatively substituted); HCDR2(e.g., SEQ ID NO: 20, SEQ ID NO: 20 wherein optionally one or two aminoacids are conservatively substituted); and HCDR3 (e.g., SEQ ID NO: 21,SEQ ID NO: 21 wherein optionally one or two amino acids areconservatively substituted)) of the monoclonal antibody sc209; and allthree light chain complementarity determining regions (LCDR1, (e.g., SEQID NO: 28 or SEQ ID NO: 28 wherein optionally one or two amino acids areconservatively substituted); LCDR2, (e.g., SEQ 113 NO: 29 or SEQ ID NO:29 wherein optionally one or two amino acids are conservativelysubstituted); and LCDR3, (e.g., SEQ ID NO: 30 or SEQ ID NO: 30 whereinoptionally one or two amino acids are conservatively substituted)) ofthe monoclonal antibody sc209. In some embodiments, the heavy chainsequence comprises human heavy chain constant region sequence of humanisotype IgG1 or IgG2.

In additional aspects, the antibody or antigen binding fragmentcomprises an amino acid sequence shown as SEQ ID NO:12, an amino acidsequence of the light chain variable region amino acid sequence of theantibody encoded by the purified DNA having ATCC Accession NumberPTA-6296, or the amino encoded by the nucleotide sequence shown asnucleic acid residues 15-719 of SEQ ID NO:11.

In other additional aspects, the antibody or antigen binding fragmentthereof comprises an amino acid sequence shown as SEQ ID NO:10, an aminoacid sequence of the heavy chain variable region amino acid sequence ofthe antibody encoded by the purified DNA having ATCC Accession NumberPTA-6296, or the amino acid sequence encoded by the nucleotide sequenceshown as nucleic acid residues 13-1440 of SEQ ID NO:9.

In still other aspects, the antibody or antigen binding fragmentcomprises a light chain variable region comprising the amino acidsequence shown as SEQ ID NO:12, or the light chain variable region aminoacid sequence of the antibody encoded by the purified DNA having ATCCAccession Number PTA-6296; and a heavy chain variable region comprisingthe amino acid sequence shown as SEQ ID NO:10, or the heavy chainvariable region amino acid sequence of the antibody encoded by thepurified DNA having ATCC Accession Number PTA-6296. In certain aspectsantibody or antigen binding fragment comprises two heavy and two lightchains.

In specific embodiments the human antibody or antigen binding fragmentis sc209, or a fragment thereof.

Provided antibodies that bind the extracellular domain of OV064, induceinternalization of OV064, and deliver toxic payload include chimericantibodies or humanized antibodies, and antigen-binding fragments of theforegoing. Such chimeric or humanized antibodies and antigen bindingfragments thereof are useful in the immunoconjugates, compositions, andmethods of treatment and diagnosis provided herein.

In some embodiments, the antibody is mAb 8G5, mAb 4G10, mAb 3A4, or mAb2F3 or an antigen-binding fragment of mAb 8G5, mAb 4G10, mAb 3A4, or mAb2F3. Also provided is a chimeric or humanized mAb 8G5 or an antigenbinding fragment thereof, a chimeric or humanized mAb 4G10 or an antigenbinding fragment thereof, a chimeric or humanized mAb 3A4 or an antigenbinding fragment thereof, or a chimeric or humanized mAb 2F, or anantigen binding fragment thereof.

Provided antibodies that bind the extracellular domain of OV064, induceinternalization of OV064, and deliver toxic payload include humanantibodies, and derivatives thereof. Such human antibodies and antigenbinding fragments thereof are useful in the immunoconjugates,compositions, and methods of treatment and diagnosis provided herein.

In some embodiments, the antibody is mAb sc77, mAb sc189, or mAb sc209or an antigen binding fragment of mAb sc77, an antigen binding fragmentof mAb sc 189, or an antigen binding fragment of mAb sc209.

In certain aspects, the methods of treatment of cancer include treatmentof a subject with an immunoconjugate of the invention as individualtherapy. In other aspects, the immunoconjugate can be used incombination with one or more other therapies. For example, theimmunoconjugate may be combined with one or more additionalchemotherapeutic agent(s) for combination therapy. Chemotherapeuticagents for combination therapy may suppress tumor growth, or kill tumorcells. Thus, methods of the invention comprise the methods of treatmentof cancer comprising administering an immunoconjugate of the inventionas described herein, and in combination with an additionalchemotherapeutic agent. The methods comprising administration of anadditional chemotherapeutic include administration of the additionalagent prior to, concurrent with, or following administration of theimmunoconjugate.

In certain embodiments, the provided methods are useful for treatment ofa cancer which is a solid tumor, soft tissue tumor, liquid tumor, or ametastastic lesion of any of the foregoing. For example, the cancer maybe ovarian cancer, breast cancer, lung cancer, pancreatic cancer orendometrial cancer.

In specific embodiments, the cancer is ovarian cancer, and the methodsmay be used for example, for treatment of ovarian adenocarcinoma, clearcell ovarian carcinoma, or serous ovarian carcinoma. Additionally, themethods may be useful for treatment of metastatic ovarian cancerinvolving a uterine, pelvic, extra-pelvic peritoneal, non-peritoneal orlymph node metastasis.

In specific embodiments, the cancer is lung cancer, and the methods maybe used for example, for treatment of squamous cell carcinoma, ornon-small-cell lung cancer.

In specific embodiments, the cancer is breast cancer, and the methodsmay be used for example, for treatment of early stage or late stagebreast cancers, or different classification of breast cancer patients,including stage 0 breast cancer patients, stage I breast cancerpatients, stage IIA breast cancer patients, stage IIB breast cancerpatients, stage IIIA breast cancer patients, stage IIIB breast cancerpatients, stage IV breast cancer patients, grade I breast cancerpatients, grade II breast cancer patients, grade III breast cancerpatients, malignant breast cancer patients, ductal carcinoma breastcancer patients, and lobular carcinoma breast cancer patients.

The invention also relates to fusion proteins comprising an antibody orportion thereof (e.g., heavy chain, light chain, variable region) of theinvention and a non-immunoglobulin moiety.

The invention also relates to immunoconjugates comprising an antibody oran antigen-binding fragment of the invention. In some aspects theimmunoconjugate comprises a cytotoxic moiety. For example, the cytotoxicmoiety can comprise a radioisotope (e.g, a radioactive ion), atherapeutic agent (e.g., a chemotherapeutic agent, an antimetabolite, analkylating agent, an anthracycline, an antibiotic, an anti-mitoticagent, a biological response modifier (e.g., a cytokine (e.g., aninterleukin, an interferon, a tumor necrosis factor), a growth factor(e.g., a neurotrophic factor)), or a tumor activated pro-drug (e.g., anenzyme and/or enzyme activated compound). When the cytotoxic moiety ofthe immunoconjugate is a therapeutic agent, the agent can comprise amaytansine, an auristatin, a dolastatin, a duocarmycin, a cryptophycin,a taxol, a DNA alkylating agent, a calicheamicin, or a derivative of anyof the foregoing. In some aspects the cytotoxic moiety comprises amaytansine selected from DM1 or DM4. In other aspects theimmunoconjugate comprises a detectable label moiety.

The invention further relates to liposome compositions comprising theantibodies, antigen-binding fragments, or immunoconjugates thereof ofthe invention. In some embodiments, the liposome is coated with antibodyor antigen binding fragment. In additional embodiments, the liposome isfilled with a cytotoxic or a cytostatic agent. In some aspects, theliposome is filled with a cytotoxin selected from the group consistingof a maytansine, an auristatin, a dolastatin, a duocarmycin, acryptophycin, a taxol, a DNA alkylating agent, or a calicheamicin, or aderivative of the foregoing. In another embodiment the liposome isfilled with nucleic acid sequence comprising RNA interference moleculeswhich are capable of diminishing OV064 expression.

The invention further relates to an antibody, an antigen-bindingfragment of an antibody, an antigen binding fragment of an antibody, afusion protein or an immuno-conjugate of the invention as describedherein for use in therapy or diagnosis of a cancer (e.g., ovariancancer, breast cancer, lung cancer, pancreatic cancer and endometrialcancer). Additionally the invention relates to the use of an antibody,an antigen-binding fragment of an antibody, a fusion protein or animmuno-conjugate of the invention for the manufacture of a medicamentfor the treatment of a cancer (e.g., ovarian cancer, breast cancer, lungcancer, pancreatic cancer and endometrial cancer).

Still further the invention relates to pharmaceutical compositionscomprising an antibody or antigen-binding fragment of the invention anda physiological acceptable carrier. Additionally encompassed in theinvention are pharmaceutical compositions comprising immunoconjugates,and/or liposome compositions of the invention and a physiologicallyacceptable carrier.

The invention also relates to a method for detecting an OV064 expressingcell, or tumors expressing OV064, comprising contacting a compositioncomprising a cell or a tumor with a detectably labeled antibody orantigen-binding fragment thereof of the invention under conditions whichallow interaction of the anti-OV064 antibody and the OV064 protein, anddetecting formation of a complex between antibody or antigen bindingfragment and the OV064 protein in the sample in order to detect thepresence of the OV064 expressing cell or tumor expressing OV064 in thesample.

In one embodiment, methods for detecting the presence of tumor cellsexpressing OV064 can be performed in vivo, wherein a detectably labeledanti-OV064 antibody or antigen binding fragment of the invention isadministered to a subject under conditions that allow interaction of theOV064 antibody or antigen binding fragment and the OV064 protein tooccur; followed by detection of formation of a complex of OV064 andantibody or antigen binding fragment to thereby detect the presence oftumor cells expressing OV064 in vivo. The methods of detection, whetherin vitro or in vivo, are useful for diagnosing or staging a cancerousdisorder. In some embodiments, the disorder for diagnosis or staging isovarian cancer, breast cancer, lung cancer, pancreatic cancer orendometrial cancer.

Any anti-OV064 antibody or antigen binding fragment of the inventionprovided herein may be useful in the diagnostic methods. For example,the antibody or antigen binding fragment can be an antibody or antigenbinding fragment that competes with any monoclonal antibody selectedfrom sc77, sc189, sc209, 3A4, 2F3, 4G10, and/or 8G5, which detects OV064expressed on cells. The anti-OV064 antibody or antigen binding fragmentthereof of the invention used in the in vivo and in vitro diagnosticmethods can be directly or indirectly labeled with a detectablesubstance to facilitate detection of the bound or unbound binding agent.Suitable detectable substances include various biologically activeenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, paramagnetic (e.g., nuclear magnetic resonance active)materials, and radioactive materials. In some embodiments, theanti-OV064 antibody or fragment thereof is coupled to a radioactive ion,e.g., indium (¹¹¹In), iodine (¹³¹I or ¹²⁵I), yttrium (⁹⁰Y), lutetium(¹⁷⁷Lu), actinium (²²⁵Ac), bismuth (²¹²Bi or ²¹³Bi), sulfur (³⁵S),carbon (¹⁴C), tritium (³H), rhodium (¹⁸⁸Rh), technetium (⁹⁹mTc),praseodymium, or phosphorous (³²P).

The invention also relates to isolated and/or recombinant nucleic acidsencoding the antibodies, antigen-binding fragments, heavy chains, lightchains and portions of the heavy chains and light chains of theantibodies described herein, and to expression constructs or vectorscomprising the expression constructs. In specific embodiments theinvention includes plasmid Ov64sc077 (ATCC Accession No. PTA-6294),plasmid Ov64sc 189 (ATCC Accession No. PTA-6295) or plasmid Ov64sc209(ATCC Accession No. PTA-6296). Still further, the invention encompassesantibodies produced by host cells expressing the antibodies encoded byplasmid Ov64sc077 (ATCC Accession No. PTA-6294), plasmid Ov64sc189 (ATCCAccession No. PTA-6295) or plasmid Ov64sc209 (ATCC Accession No.PTA-6296).

For example, in one embodiment is provided an expression vectorcomprising a gene encoding a humanized antibody light chain, comprisinga nucleotide sequence encoding a complementarity determining regionsequence derived from a light chain of a murine antibody (e.g., 4G10,3A4, 2F3, 8G5), and a framework region derived from a light chain ofhuman origin. In another embodiment is provided an expression vectorcomprising a gene encoding a humanized antibody heavy chain, comprisinga nucleotide sequence encoding a complementarity determining regionderived from a heavy chain of a murine antibody (e.g., 4G10, 3A4, 2F3,8G5), and a framework region derived from a heavy chain of human origin.An additional embodiment provides a host cell comprising the foregoingexpression vectors, expressing heavy and light chain antibodies, and astill further embodiment provides a method of producing a humanizedantibody comprising maintaining the host cell under conditionsappropriate for expression of a humanized immunoglobulin, wherebyhumanized immunoglobulin chains are expressed and a humanized antibodyis produced. The host cell can be a eukaryotic cell, e.g., a mammaliancell, an insect cell, a yeast cell, or a prokaryotic cell, e.g., E.coli. For example, the mammalian cell can be a cultured cell or a cellline. Exemplary mammalian cells include lymphocytic cell lines (e.g.,NS0), Chinese hamster ovary cells (CHO), COS cells. Additionally cellsinclude oocyte cells, and cells from a transgenic animal, e.g., mammaryepithelial cell. For example, nucleic acids encoding an antibody or amodified antibody or an antigen binding fragment thereof describedherein can be expressed in a transgenic animal.

In another embodiment is provided an expression vector comprising a geneencoding a human antibody light chain, comprising a nucleotide sequenceencoding one, two, or three complementarity determining region sequencesfrom a light chain of a human antibody (e.g., sc77, sc 189, or sc209),and a framework region derived from a light chain of human origin. Forexample, provided expression vectors include expression vectors encodinga human antibody light chain comprising all three complementaritydetermining regions from a light chain of human antibody (e.g., sc77,sc189, or sc209). In another embodiment is provided an expression vectorcomprising a gene encoding a human antibody heavy chain, comprising anucleotide sequence encoding one, two, or three complementaritydetermining regions from a heavy chain of a human antibody (e.g., sc77,sc189, or sc209), and a framework region derived from a heavy chain ofhuman origin. Additionally, provided expression vectors includeexpression vectors encoding a human antibody heavy chain comprising allthree complementarity determining regions from a heavy chain of humanantibody (e.g., sc77, sc189, or sc209). An additional embodimentprovides a host cell comprising the foregoing expression vectorsencoding heavy and light chain antibody sequences. The host cell can bea eukaryotic cell, e.g., a mammalian cell, an insect cell, a yeast cell,or a prokaryotic cell, e.g., E. coli. For example, the mammalian cellcan be a cultured cell or a cell line. Exemplary mammalian cells includelymphocytic cell lines (e.g., NS0), Chinese hamster ovary cells (CHO),COS cells. Additionally cells include oocyte cells, and cells from atransgenic animal, e.g., mammary epithelial cell. For example, nucleicacids encoding an antibody or a modified antibody or an antigen bindingfragment thereof described herein can be expressed in a transgenicanimal.

A still further embodiment provides a method of producing a humanantibody comprising maintaining the host cell under conditionsappropriate for expression of a human immunoglobulin, whereby humanimmunoglobulin chains are expressed and a human antibody is produced.For example, methods of expression of human antibodies include use ofhost cells wherein a first recombinant nucleic acid molecule encoding ahuman antibody light chain and a second recombinant nucleic acidmolecule encoding a human antibody heavy chain are comprised in a singleexpression vector

The invention also relates to a host cell that comprises a nucleic acidof the invention. In specific embodiments, the invention is Hybridoma8G5 (ATCC Accession No. PTA-6403), Hybridoma 4G10 (ATCC Accession No.PTA-6402), Hybridoma 3A4 (ATCC Accession No. PTA-6401), or Hybridoma 2F3(ATCC Accession No. PTA-6400). Still further, the invention encompassesantibodies produced by Hybridoma 8G5 (ATCC Accession No. PTA-6403),Hybridoma 4G10 (ATCC Accession No. PTA-6402), Hybridoma 3A4 (ATCCAccession No. PTA-6401), or Hybridoma 2F3 (ATCC Accession No. PTA-6400).

We have found that certain antibodies to the Ov064 (also known as B7H4,B7x, B7S1) protein rapidly internalize and can bring toxic payloads oftoxin (e.g., maytansines, e.g., DM1 and DM4) into Ov064 expressingcells, leading to cell death. Ov064 is expressed on >90% of ovariancancer tissues as well as breast and lung cancer tissue samples. SeeInternational Patent Publication No. WO200012758, published Mar. 9,2000; International Patent Publication No. WO200036107, published Jun.22, 2000; U.S. Patent Application Publication No. US20030165504,published Sep. 4, 2003; and U.S. Patent Application Publication No.US20030091580, published May 15, 2003. As a result, we concluded toxinconjugated antibodies to OV064 have therapeutic utility in cancers whereOV064 is expressed (e.g., breast cancer, ovarian cancer). Ov064, alsoknown as B7-H4, B7x, and B7S1, a 282 amino acid protein, has beendescribed as a member of the B7-family of lymphocyte co-inhibitoryproteins (See, Prasad et al., Immunity 18: 863-873 (2003); Sica et al.,Immunity 18: 849-861 (2003); Zang et al., Proc Natl Acad Sci USA 100:10388-10392 (2003); GenBank Accession No. NMO24626, each of which areincorporated herein by reference). It has also been demonstrated thatOv064 is a ligand for BTLA, a co-inhibitory receptor which shareshomology with CTLA4 and CD28 co-stimulatory receptors. Ov064 has beenshown to inhibit T-cell activation (Prasad et al., Immunity 18: 863-873(2003); Sica et al., Immunity 18: 849-861 (2003); Zang et al., Proc NatlAcad Sci USA 100: 10388-10392 (2003)). It is also possible that tumorcells express this antigen to avoid being recognized by the immunesystem. Thus, anti-Ov064 antibodies may act in certain aspects in asimilar manner as that found for anti-CTLA4 antibodies (Egen et al., NatImmunol 3: 611-618 (2002). An antibody to Ov064 therefore could possiblyexert a dual effect in patients with cancer: anti-Ov064 antibodies maydeliver a toxic payload to tumor cells expressing OV064, as well asdiminish the immunosuppressive effects of Ov064, thereby enhancing tumorcell killing via direct delivery of toxin, as well as activated immunecells. We have found that only a subset of antibodies which bind OV064are efficacious in delivery to cells of toxic payloads sufficient toinduce cell killing. This effect does not appear to relate directly toaffinity of binding to OV064, rather a subset of antibodies whichcompete with the monoclonal antibodies and human monoclonal antibodiesprovided herein are preferred for use in the methods described. We havefound an anti-OV064 antibody which binds to an epitope located withinthe region of about amino acids 133-257 of SEQ ID NO:44 is capable ofdelivering toxic payload to a cell expressing OV064. For example, ananti-OV064 antibody which binds to an epitope located wholly orpartially within the region of about amino acids 167-176 or about aminoacids 177-181 of human OV064 (SEQ ID NO:44) is capable of delivery oftoxic payload. In another example, an anti-OV064 antibody which binds toan epitope wholly or partially within the region of about amino acids238-257 of human OV064 (SEQ ID NO:44) is capable of delivery of toxicpayload. In contrast, an anti-OV064 antibody which binds to an epitopelocated within the region of amino acids 32-133 of SEQ ID NO:44 wasfound not capable of delivery of toxic payload to cells expressingOV064. For example, an antibody which binds an epitope located wholly orpartially within the region of about amino acids 67-76 of human OV064(SEQ ID NO:44) was incapable of delivery of toxic payload.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention have the meanings that arecommonly understood by those of ordinary skill in the art. Generally,nomenclature utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation and transfection (e.g., electroporation, lipofection).Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications or as commonly accomplished in the artor as described herein. The foregoing techniques and procedures aregenerally performed according to conventional methods well known in theart and as described in various general and more specific referencesthat are cited and discussed throughout the present specification. Seee.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (3rd ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000)),See generally, Harlow, E. and Lane, D. (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.The nomenclatures utilized in connection with, and the laboratoryprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art. Standard techniquesare used for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients.Furthermore, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.

The instant invention is most clearly understood with reference to thefollowing definitions, as well as additional definitions as set forththroughout the description:

As used herein, the term “antibody” “antibody peptide(s)” or“immunoglobulin” refers to single chain, two-chain, and multi-chainproteins and glycoproteins that belong to the classes of polyclonal,monoclonal, chimeric and human or humanized immunoglobulin proteins. Theterm “antibody” also includes synthetic and genetically engineeredvariants thereof.

As used herein, the term “antibody fragment” or “antigen bindingfragment” of an antibody refers to Fab, Fab′, F(ab′)₂, and Fv fragments,single chain antibodies, functional heavy chain antibodies (nobodies),as well as any portion of an antibody having specificity toward at leastone desired epitope, that competes with the intact antibody for specificbinding (e.g., an isolated portion of a complementarity determiningregion having sufficient framework sequences so as to bind specificallyto an epitope). Antigen binding fragments can be produced by recombinanttechniques, or by enzymatic or chemical cleavage of an intact antibody.

As used herein, the term “human antibody” refers to an antibody thatpossesses a sequence that is derived from a human germ-lineimmunoglobulin sequence, such as antibodies derived from transgenic micehaving human immunoglobulin genes (e.g., XENOMOUSE™ geneticallyengineered mice (Abgenix)), human phage display libraries, or human Bcells.

As used herein, the term “humanized antibody” refers to an antibody thatis derived from a non-human antibody (e.g., murine) that retains orsubstantially retains the antigen-binding properties of the parentantibody but is less immunogenic in humans. Humanized as used herein isintended to include deimmunized antibodies.

The term “modified” antibody, as used herein, refers to antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial antibody library, antibodies isolated from an animal(e.g., a mouse) that is transgenic for human immunoglobulin genes orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such modified antibodies include humanized, CDR grafted,chimeric, in vitro generated (e.g., by phage display) antibodies, andmay optionally include variable or constant regions derived from humangermline immunoglobulin sequences or human immunoglobulin genes orantibodies which have been prepared, expressed, created or isolated byany means that involves splicing of human immunoglobulin gene sequencesto alternative immunoglobulin sequences.

The term “monospecific antibody” refers to an antibody that displays asingle binding specificity and affinity for a particular target, e.g.,epitope. This term includes a “monoclonal antibody” or “monoclonalantibody composition,” which as used herein refer to a preparation ofantibodies or fragments thereof of single molecular composition.

The term “bispecific antibody” or “bifunctional antibody” refers to anantibody that displays dual binding specificity for two epitopes, whereeach binding site differs and recognizes a different epitope.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

The term “anti-cancer agent” or “chemotherapeutic agent” is used hereinto refer to agents that have the functional property of inhibiting adevelopment or progression of a neoplasm in a human, particularly amalignant (cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, orleukemia. Inhibition of metastasis is frequently a property ofanti-cancer or chemotherapeutic agents. A chemotherapeutic agent may bea cytotoxic or cytostatic agent. A cytostatic agent refers to an agentwhich inhibits or suppresses cell growth and/or multiplication of cells.

“Cytotoxic agents” refer to compounds which cause cell death primarilyby interfering directly with the cell's functioning or inhibit orinterfere with cell myosis, including, but not limited to, alkylatingagents, tumor necrosis factors, intercalators, microtubulin inhibitors,and topoisomerase inhibitors. A toxic payload as used herein refers to asufficient amount of cytotoxic agent which, when delivered to a cellresults in cell death. A toxic payload may include a sufficient amountof immunoconjugate comprising a cytotoxic agent, wherein theimmunoconjugate comprises an antibody or antigen binding fragment of theinvention. A toxic payload may also include a sufficient amount of animmunoconjugate comprising a cytotoxic agent, wherein theimmunoconjugate comprises a secondary antibody or antigen bindingfragment thereof which recognizes and binds an antibody or antigenbinding fragment of the invention.

As used herein the phrase, a sequence “derived from” or “specific for adesignated sequence” refers to a sequence that comprises a contiguoussequence of approximately at least 6 nucleotides or at least 2 aminoacids, preferably at least about 9 nucleotides or at least 3 aminoacids, more preferably at least about 10-12 nucleotides or 4 aminoacids, and even more preferably at least about 15-21 nucleotides or 5-7amino acids corresponding, i.e., identical or complementary to, a regionof the designated sequence. In certain embodiments, the sequencecomprises all of a designated nucleotide or amino acid sequence. Thesequence may be complementary (in the event a polynucleotide sequence)or identical to a sequence that is unique to a particular sequence asdetermined by techniques known in the art. Regions from which sequencesmay be derived, include but are not limited to, regions encodingspecific epitopes, regions encoding complementarity determining regions,regions encoding framework sequences, regions encoding constant domainregions, regions encoding variable domain regions, as well asnon-translated and/or non-transcribed regions. The derived sequence willnot necessarily be derived physically from the sequence of interestunder study, but may be generated in any manner, including, but notlimited to, chemical synthesis, replication, reverse transcription ortranscription, that is based on the information provided by the sequenceof bases in the region(s) from which the polynucleotide is derived. Assuch, it may represent either a sense or an antisense orientation of theoriginal polynucleotide. In addition, combinations of regionscorresponding to that of the designated sequence may be modified orcombined in ways known in the art to be consistent with the intendeduse. For example, a sequence may comprise two or more contiguoussequences which each comprise part of a designated sequence, and areinterrupted with a region which is not identical to the designatedsequence is intended to represent a sequence derived from the designatedsequence.

As used herein, the phrase “encoded by” refers to a nucleic acidsequence that codes for a polypeptide sequence, wherein the polypeptidesequence or a portion thereof contains an amino acid sequence of atleast 3 to 5 amino acids, more preferably at least 8 to 10 amino acids,and even more preferably at least 15 to 20 amino acids from apolypeptide encoded by the nucleic acid sequence. The nucleic acidsequences of the present invention which code for antibodies can beselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11. Also encompassed arepolypeptide sequences that are immunologically identifiable with apolypeptide encoded by the sequence. Thus, a “polypeptide,” “protein” or“amino acid” sequence has at least about 70%, 75%, 80%, 85%, 90%, 95% ormore identity to the antibodies of the present invention. Further, theantibodies of the present invention may have at least about 60%, 70%,75%, 80%, 85%, 90% or 95% similarity to a polypeptide or amino sequencesof the antibodies of the present invention. The amino acid sequences ofthe antibodies of the present invention can be selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

As used herein, the term “substantially identical” (or “substantiallyhomologous”) is used herein to refer to a first amino acid or nucleotidesequence that contains a sufficient number of identical or equivalent(e.g., with a similar side chain, e.g., conserved amino acidsubstitutions) amino acid residues or nucleotides to a second amino acidor nucleotide sequence such that the first and second amino acid ornucleotide sequences have similar activities. In the case of antibodies,the second antibody has the same specificity and has at least 50% of theaffinity of the same.

Calculations of “homology” between two sequences can be performed asfollows. The sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The length of a reference sequence aligned for comparison purposes is atleast 30%, 40%, or 50%, preferably at least 60%, and more preferably atleast 70%, 80%, 90%, 100% of the length of the reference sequence. Theamino acid residues or nucleotides at corresponding amino acid positionsor nucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. The percent homology between two amino acid sequences can bedetermined using any method known in the art. For example, the Needlemanand Wunsch, J. Mol. Biol. 48:444-453 (1970), algorithm which has beenincorporated into the GAP program in the GCG software package, usingeither a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16,14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent homology between two nucleotide sequences can also be determinedusing the GAP program in the GCG software package, using a NWSgapdna.CMPmatrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of1, 2, 3, 4, 5, or 6. An exemplary set of parameters for determination ofhomology are a Blossum 62 scoring matrix with a gap penalty of 12, a gapextend penalty of 4, and a frameshift gap penalty of 5.

As used herein, the term “hybridizes under stringent conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. Specific hybridizationconditions referred to herein are as follows: 1) low stringencyhybridization conditions in 6× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at50° C. (the temperature of the washes can be increased to 55° C. for lowstringency conditions); 2) medium stringency hybridization conditions in6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC atabout 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C.; and 4) very high stringency hybridization conditions are 0.5M sodiumphosphate, 7% SDS at 65° C., followed by one or more washes at 0.2×SSC,1% SDS at 65° C. Very high stringency conditions (4) are the preferredconditions and the ones that should be used unless otherwise specified.

It is understood that the antibodies and antigen binding fragmentthereof of the invention may have additional conservative ornon-essential amino acid substitutions, which do not have a substantialeffect on the polypeptide functions. Whether or not a particularsubstitution will be tolerated, i.e., will not adversely affect desiredbiological properties, such as binding activity can be determined asdescribed in Bowie, J U et al. Science 247:1306-1310 (1990). A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolarside chains (e.g., glycine, alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of the binding agent, e.g., the antibody,without abolishing or more preferably, without substantially altering abiological activity, whereas an “essential” amino acid residue resultsin such a change.

As used herein, the term “isolated” refers to material that is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotide could be part of a vector and/or such polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat the vector or composition is not part of its natural environment.

As used herein, the term “replicon” refers to any genetic element, suchas a plasmid, a chromosome or a virus, that behaves as an autonomousunit of polynucleotide replication within a cell.

As used herein, the term “operably linked” refers to a situation whereinthe components described are in a relationship permitting them tofunction in their intended manner. Thus, for example, a control sequence“operably linked” to a coding sequence is ligated in such a manner thatexpression of the coding sequence is achieved under conditionscompatible with the control sequence.

As used herein, the term “vector” refers to a replicon in which anotherpolynucleotide segment is attached, such as to bring about thereplication and/or expression of the attached segment.

As used herein, the term “control sequence” refers to a polynucleotidesequence that is necessary to effect the expression of a coding sequenceto which it is ligated. The nature of such control sequences differsdepending upon the host organism. In prokaryotes, such control sequencesgenerally include a promoter, a ribosomal binding site and terminatorsand, in some instances, enhancers. The term “control sequence” thus isintended to include at a minimum all components whose presence isnecessary for expression, and also may include additional componentswhose presence is advantageous, for example, leader sequences.

As used herein, the term “purified product” refers to a preparation ofthe product which has been isolated from the cellular constituents withwhich the product is normally associated and from other types of cellsthat may be present in the sample of interest.

As used herein, the term “epitope” refers to any protein determinatecapable of binding specifically to an antibody or T-cell receptors.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic as described in more detail herein. Various methodsof labeling polypeptides and glycoproteins are known in the art and maybe used. Examples of labels for polypeptides include, but are notlimited to, the following: radioisotopes or radionuclides (e.g., ³H,¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g.,FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g.,horseradish peroxidase, .beta.-galactosidase, luciferase, alkalinephosphatase), chemiluminescent, biotinyl groups, predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, epitope tags). In some embodiments, labels are attachedby spacer arms of various lengths to reduce potential steric hindrance.

As used herein, “specific binding” “bind(s) specifically” or “bindingspecificity” refers to the property of the antibody to: (1) to bind toOV064, e.g., human OV064 protein, with an affinity of at least 1×10⁷M⁻¹,and (2) preferentially bind to OV064, e.g., human OV064 protein, with anaffinity that is at least two-fold, 50-fold, 100-fold, 1000-fold, ormore greater than its affinity for binding to a non-specific antigen(e.g., BSA, casein) other than OV064.

As used herein, the term “treat” or “treatment” is defined as theapplication or administration of an anti-OV064 antibody or antigenbinding fragment thereof to a subject, e.g., a patient, or applicationor administration to an isolated tissue or cell from a subject, e.g., apatient, which is returned to the subject. The anti-OV064 antibody orantigen binding fragment thereof, can be administered alone or incombination with, a second agent. The subject can be a patient (e.g., ahuman patient, a veterinary patient) having a cancer (e.g., ovariancancer, breast cancer, lung cancer, pancreatic cancer and endometrialcancer)), a symptom of a cancer in which at least some of the cellsexpress OV064 (e.g., ovarian cancer, breast cancer, lung cancer,pancreatic cancer and endometrial cancer), or a predisposition toward acancer in which at least some of the cells express OV064 (e.g., ovariancancer, breast cancer, lung cancer, pancreatic cancer and endometrialcancer). The treatment can be to cure, heal, alleviate, relieve, alter,remedy, ameliorate, palliate, improve or affect the disorder, thesymptoms of the disorder or the predisposition toward the cancer. Whilenot wishing to be bound by theory treating is believed to cause theinhibition, ablation, or killing of a cell in vitro or in vivo, orotherwise reducing capacity of a cell, e.g., an aberrant cell, tomediate a disorder, e.g., a disorder as described herein (e.g., acancer).

As used herein, an amount of an anti-OV064 antibody “effective” or“sufficient” to treat a disorder, or a “therapeutically effectiveamount” or “therapeutically sufficient amount” refers to an amount ofthe antibody which is effective, upon single or multiple doseadministration to a subject, in treating a cell, e.g., cancer cell(e.g., an OV064-expressing tumor cell), or in prolonging curing,alleviating, relieving or improving a subject with a disorder asdescribed herein beyond that expected in the absence of such treatment.As used herein, “inhibiting the growth” of the tumor or cancer refers toslowing, interrupting, arresting or stopping its growth and metastasesand does not necessarily indicate a total elimination of the tumorgrowth.

As used herein, “OV064,” also known as “B7H4”, “B7x”, “B7S1,” “Ovr110”protein refers to mammalian OV064, preferably human OV0664 protein.Human OV064 includes the protein product encoded by the nucleic acidsequence of OV064 shown in SEQ ID NO: 43; the amino acid sequence ofOV064 is shown in SEQ ID NO: 44. The transcript encodes a proteinproduct of 282 amino acids, and is described in Genbank accession no.:AY280972, submitted Apr. 22, 2003, and characterized as a B7-like memberof the immunoglobulin superfamily of proteins, believed to play acritical role in antigen specific immune responses. OV064 has beencharacterized as a protein involved in immune responses, as well ascancers, including ovarian, breast, lung, endometrial, and pancreaticcancers. See also, Prasad et al., Immunity 18: 863-873 (2003); Sica etal., Immunity 18: 849-861 (2003); Zang et al., Proc Natl Acad Sci USA100: 10388-10392 (2003); also International Patent Publication No.WO9963088, published Dec. 9, 1999; International Patent Publication No.WO200012758, published Mar. 9, 2000; International Publication No.WO200036107, published Jun. 22, 2000; International Publication No.WO200140269, published Jun. 7, 2001; International Publication No.WO200206317, published Jan. 24, 2002; International Publication No.WO0194641, published Dec. 13, 2001; International Publication No.WO0202624, published Jan. 10, 2002; International Publication No.WO02010187, published Feb. 7, 2002; International Publication No.WO03000012, published Jan. 3, 2003; International Publication No.WO04000221, published Dec. 31, 2003; International Publication No.WO040101756, published Nov. 25, 2004; and International Publication No.WO04113500, published Dec. 29, 2004. Accordingly, the term “human OV064”refers to protein product which has or is homologous to (e.g., at leastabout 85%, 90%, 95% identical to) an amino acid sequence as shown in SEQID NO: 44, or which is encoded by (a) a human OV064 nucleic acidsequence (e.g., SEQ ID NO: 43); (b) a nucleic acid sequence degenerateto a naturally occurring human OV064 sequence; (c) a nucleic acidsequence homologous to (e.g., at least about 85%, 90%, 95% identical to)the naturally occurring human OV064 nucleic acid sequence; or (d) anucleic acid sequence that hybridizes to one of the foregoing nucleicacid sequences under stringent conditions, e.g., highly stringentconditions. The OV064 target is a member of the co-inhibitory cellsurface molecules that can down regulate or inhibit an immune response(Chen, Nat Rev Immunol 4: 336-347 (2004)). It is generally known asB7-H4, B7x or B7S1 in the literature, and is believed to interact withBTLA (Choi et al., J Immunol 171: 4650-4654 (2003); Prasad et al.,Immunity 18: 863-873 (2003); Sica et al., Immunity 18: 849-861 (2003);Zang et al., Proc Natl Acad Sci USA 100: 10388-10392 (2003). Further,OV064 is a cell surface protein found in greater than 90% of ovariancancers and 25-30% of lung and breast cancer patient samples (See Choiet al., J Immunol 171: 4650-4654 (2003)). Antibody therapeutics directedto Ov064 can be used alone in unconjugated form to thereby inhibit theOV064-expressing cancerous cells by, e.g., bypass tumor induced immunesuppression and promote T-cell mediated killing of tumor cells.

An “anti-OV064 antibody” is an antibody that interacts with (e.g., bindsto) OV064, preferably human OV064 protein. The provided anti-OV064antibodies or antigen binding fragments thereof interact with, e.g.,binds to, the extracellular domain of OV064, e.g., the extracellulardomain of human OV064 located at about amino acids 32-282 of humanOV064. In one embodiment, the anti-OV064 antibody or antigen bindingfragment thereof binds all or part of the epitope of an antibodydescribed herein, e.g., sc77, sc189, sc209, 4G10, 3A4, 2F3 and/or 8G5.The anti-OV064 antibody can inhibit, e.g., competitively inhibit, thebinding of an antibody described herein, e.g., sc77, sc189, sc209, 4G10,3A4 2F3 and/or 8G5 to human OV064. An anti-OV064 antibody may bind to anepitope, e.g., a conformational or a linear epitope, which epitope whenbound prevents binding of an antibody described herein, e.g., sc77,sc189, sc209, 4G10, 3A4, 2F3 and/or 8G5. In one embodiment, theanti-OV064 antibody binds to an epitope located within the region ofabout amino acids 133-257 of SEQ ID NO:44, or about amino acids 133-183of SEQ ID NO:44, and preferably wholly or partially within the region ofabout amino acids 167-176 or about amino acids 177-181 of human OV064(SEQ ID NO:44). In another embodiment, the anti-OV064 antibody binds toan epitope located within the region of about amino acids 133-257 of SEQID NO:44, or about amino acids 189-257 of SEQ ID NO:44, and preferablywholly or partially within the region of about amino acids 238-257 ofhuman OV064 (SEQ ID NO:44). In still another embodiment, the anti-OV064antibody binds to an epitope located within the region of about aminoacids 32-133 of SEQ ID NO:44, preferably wholly or partially within theregion of about amino acids 67-76 of human OV064 (SEQ ID NO:44).

Antibodies

The antibody structural unit is a tetramer. Each tetramer is composed oftwo identical pairs of polypeptide chains, each pair having one “light”(about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa and lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)) (incorporated by reference in its entirety). The variableregions of each light/heavy chain pair form the antibody binding site.Preferred isotypes for the anti-OV064 antibodies are IgGimmunoglobulins, which are classified into four subclasses, IgG1, IgG2,IgG3 and IgG4, having different gamma heavy chains. Most therapeuticantibodies are human, chimeric, or humanized antibodies of the IgG1type.

The variable regions of each heavy and light chain pair form the antigenbinding site. Thus, an intact IgG antibody has two binding sites whichare the same. However, bifunctional or bispecific antibodies areartificial hybrid constructs which have two different heavy/light chainpairs, resulting in two different binding sites.

The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hyper variable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987);Chothia et al. Nature 342:878-883 (1989). As used herein, CDRs arereferred to for each of the heavy (HCDR1, HCDR2, HCDR3) and light(LCDR1, LCDR2, LCDR3) chains.

Useful immunogens for the purpose of this invention include OV064 (e.g.,human OV064)-expressing cells (e.g., a tumor cell line, e.g., SKBR3cells, ZR75-1 cells, OVCAR3 cells, MDA-MB-468 cells, MCF7 cells, DLD1cells, or fresh or frozen ovarian tumor cells, recombinant cellsexpressing OV064); membrane fractions of OV064-expressing cells (e.g., aovarian tumor cell line, e.g., SKBR3 cells, or fresh or frozen ovariantumor cells, recombinant cells expressing OV064); isolated or purifiedOV064, e.g., human OV064 protein (e.g., biochemically isolated OV064, ora portion thereof, e.g., the extracellular domain of OV064, recombinantcells expressing OV064).

The antibodies of the present invention can be polyclonal antibodies,monoclonal antibodies, chimeric antibodies (See U.S. Pat. No. 6,020,153)or human or humanized antibodies or antibody fragments or derivativesthereof. Synthetic and genetically engineered variants (See U.S. Pat.No. 6,331,415) of any of the foregoing are also contemplated by thepresent invention. Monoclonal antibodies can be produced by a variety oftechniques, including conventional murine monoclonal antibodymethodology e.g., the standard somatic cell hybridization technique ofKohler and Milstein, Nature 256: 495 (1975). See generally, Harlow, E.and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. Preferably, for therapeuticapplications, the antibodies of the present invention are human orhumanized antibodies. The advantage of human or humanized antibodies isthat they potentially decrease or eliminate the immunogenicity of theantibody in a host recipient, thereby permitting an increase in thebioavailability and a reduction in the possibility of adverse immunereaction, thus potentially enabling multiple antibody administrations.

Modified antibodies include humanized, chimeric or CDR-graftedantibodies. Human anti-mouse antibody (HAMA) responses have led todevelopment of chimeric or otherwise humanized antibodies. Whilechimeric antibodies have a human constant region and a murine variableregion, it is expected that certain human anti-chimeric antibody (HACA)responses will be observed, particularly in chronic or multi-doseutilizations of the antibody. The presence of such murine or rat derivedproteins can lead to the rapid clearance of the antibodies or can leadto the generation of an immune response against the antibody by apatient. In order to avoid the utilization of murine or rat derivedantibodies, humanized antibodies where sequences are introduced to anantibody sequence to make it closer to human antibody sequence, or fullyhuman antibodies generated by the introduction of human antibodyfunction into a rodent have been developed so that the rodent wouldproduce antibodies having fully human sequences. Human antibodies avoidcertain of the problems associated with antibodies that possess murine,rabbit, or rat variable and/or constant regions.

Human Antibodies

Fully human antibodies are expected to minimize the immunogenic andallergic responses intrinsic to mouse or mouse-derivatized Mabs and thusto increase the efficacy and safety of the administered antibodies. Theuse of fully human antibodies can be expected to provide a substantialadvantage in the treatment of chronic and recurring human diseases, suchas inflammation, autoimmunity, and cancer, which require repeatedantibody administrations. Also, human antibodies can be produced usinggenetically engineered strains of animals in which the antibody geneexpression of the animal is suppressed and functionally replaced withhuman antibody gene expression.

Methods for making humanized and human antibodies are known in the art.One method for making human antibodies employs the use of transgenicanimals, such as a transgenic mouse. These transgenic animals contain asubstantial portion of the human antibody producing genome inserted intotheir own genome and the animal's own endogenous antibody production isrendered deficient in the production of antibodies. Methods for makingsuch transgenic animals are known in the art. Such transgenic animalscan be made using XENOMOUSE™ technology or by using a “minilocus”approach. Methods for making XENOMICE™ are described in U.S. Pat. Nos.6,162,963, 6,150,584, 6,114,598 and 6,075,181, which are incorporatedherein by reference. Methods for making transgenic animals using the“minilocus” approach are described in U.S. Pat. Nos. 5,545,807,5,545,806 and 5,625,825; also see International Publication No.WO93/12227, which are each incorporated herein by reference.

Using the XENOMOUSE™ technology, human antibodies can be obtained byimmunizing a XENOMOUSE™ mouse (Abgenix, Fremont, Calif.) with an antigenof interest. The lymphatic cells (such as B-cells) are recovered fromthe mice that express antibodies. These recovered cells can be fusedwith myeloid-type cell line to prepare immortal hybridoma cell lines,using standard methodology. These hybridoma cell lines can be screenedand selected to identify hybridoma cell lines that produce antibodiesspecific to the antigen of interest. Alternatively, the antibodies canbe expressed in cell lines other than hybridoma cell lines. Morespecifically, sequences encoding particular antibodies can be clonedfrom cells producing the antibodies and used for transformation of asuitable mammalian host cell. In a preferred method, spleen and/or lymphnode lymphocytes from immunized mice are isolated from the mice andplated in plaque assays as described previously in Babcook et al., ProcNatl Acad Sci USA. 93: 7843-8 (1996), which is incorporated herein byreference. Briefly, cells are plated in agar with sheep red blood cells,coated with OV064 antigen and cells secreting mAb against the OV064antigen would fix complement and lyse the red blood cells immediatelysurrounding the mAb producing cells. Cells within the cleared plaquesare lifted for sequencing of the immunoglobulin sequences and subcloninginto expression vectors. Supernatants from transiently transfected cellscontaining OV064 specific mAb were subsequently screened by ELISA andfor binding to cells by flow cytometry. We utilized these techniques inaccordance with the present invention for the preparation of antibodiesspecific to OV064. The variable sequences, or a portion thereof of theproduced human antibodies comprising complementarity determining regionswhich bind particular epitopes may be utilized for production ofmodified antibodies. For example, the variable regions of the producedantibodies may be spliced into an expression cassette for ease oftransfer of constructs, increased expression of constructs, and/orincorporation of constructs into vectors capable of expression of fulllength antibodies. Herein, we describe the production of multiplehybridoma cell lines that produce antibodies specific to OV064. Further,we provide a characterization of the antibodies produced by such celllines, including nucleotide and amino acid sequence analyses of theheavy and light chains of such antibodies, and vectors comprising thecoding sequences of the immunoglobulin chains.

Humanization and Display Technologies

As discussed above, there are advantages to producing antibodies withreduced immunogenicity. To a degree, this can be accomplished inconnection with techniques of humanization and display techniques usingappropriate libraries. It will be appreciated that murine antibodies orantibodies from other species can be humanized or primatized usingtechniques well known in the art. See e.g., Winter and Harris ImmunolToday 14:43-46 (1993) and Wright et al. Crit. Reviews in Immunol.12125-168 (1992). The antibody of interest may be engineered byrecombinant DNA techniques to substitute the CH1, CH2, CH3, hingedomains, and/or the framework domain with the corresponding humansequence (see WO 92/02190 and U.S. Pat. Nos. 5,530,101, 5,585,089,5,693,761, 5,693,792, 5,714,350, and 5,777,085). Also, the use of IgcDNA for construction of chimeric immunoglobulin genes is known in theart (Liu et al. Proc Natl Acad Sci USA. 84:3439 (1987) and J. Immunol.139:3521 (1987)). mRNA is isolated from a hybridoma or other cellproducing the antibody and used to produce cDNA: The cDNA of interestmay be amplified by the polymerase chain reaction using specific primers(U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library ismade and screened to isolate the sequence of interest. The DNA sequenceencoding the variable region of the antibody is then fused to humanconstant region sequences. The sequences of human constant regions genesmay be found in Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, N.I.H. publication no. 91-3242. Human C regiongenes are readily available from known clones. The choice of isotypewill be guided by the desired effector functions, such as complementfixation, or activity in antibody-dependent cellular cytotoxicity.Isotypes can be IgG1, IgG2, IgG3 or IgG4. Preferred isotypes forantibodies of the invention are IgG1 and IgG2. Either of the human lightchain constant regions, kappa or lambda, may be used. The chimeric,humanized antibody is then expressed by conventional methods.

Humanized antibodies can also be made using a CDR-grafted approach.Techniques of generation of such humanized antibodies are well known inthe art. Generally, humanized antibodies are produced by obtainingnucleic acid sequences that encode the variable heavy and variable lightsequences of an antibody that binds to OV064, identifying thecomplementary determining region or “CDR” in the variable heavy andvariable light sequences and grafting the CDR nucleic acid sequences onto human framework nucleic acid sequences. (See, for example, U.S. Pat.Nos. 4,816,567 and 5,225,539, which are incorporated by reference). Thelocation of the CDRs and framework residues can be determined (see,Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242, and Chothia, C. et al. J. Mol. Biol.196:901-917 (1987), which are incorporated herein by reference). Thehuman framework that is selected is one that is suitable for in vivoadministration, meaning that it does not exhibit immunogenicity. Forexample, such a determination can be made by prior experience with invivo usage of such antibodies and studies of amino acid similarities.

Once the CDRs and FRs of the cloned antibody that are to be humanizedare identified, the amino acid sequences encoding the CDRs areidentified and the corresponding nucleic acid sequences grafted on toselected human FRs. This can be done using known primers and linkers,the selection of which are known in the art. All of the CDRs of aparticular human antibody may be replaced with at least a portion of anon-human CDR or only some of the CDRs may be replaced with non-humanCDRs. It is only necessary to replace the number of CDRs required forbinding of the humanized antibody to a predetermined antigen. After theCDRs are grafted onto selected human FRs, the resulting “humanized”variable heavy and variable light sequences are expressed to produce ahumanized Fv or humanized antibody that binds to OV064. Typically, thehumanized variable heavy and light sequences are expressed as a fusionprotein with human constant domain sequences so an intact antibody thatbinds to OV064 is obtained. However, a humanized Fv antibody can beproduced that does not contain the constant sequences.

Also within the scope of the invention are humanized antibodies in whichspecific amino acids have been substituted, deleted or added. Inparticular, humanized antibodies have amino acid substitutions in theframework region, such as to improve binding to the antigen. Forexample, a selected, small number of acceptor framework residues of thehumanized immunoglobulin chain can be replaced by the correspondingdonor amino acids. Locations of the substitutions include amino acidresidues adjacent to the CDR, or which are capable of interacting with aCDR (see e.g., U.S. Pat. No. 5,585,089). The acceptor framework can be amature human antibody framework sequence or a consensus sequence. Asused herein, the term “consensus sequence” refers to the sequence formedfrom the most frequently in a region among related family members.

Other techniques for humanizing antibodies are described in Padlan etal. EP 519596 A1, published on Dec. 23, 1992.

The anti-OV064 antibody, or antigen fragment thereof, includes otherhumanized antibodies which may also be modified by specific deletion ofhuman T cell epitopes or “deimmunization” by the methods disclosed in WO98/52976 and WO 00/34317, the contents of which are incorporated hereinby reference. Briefly, the murine heavy and light chain variable regionsof an anti-OV064 antibody can be analyzed for peptides that bind to MHCClass II; these peptides represent potential T-cell epitopes. Fordetection of potential T-cell epitopes, a computer modelling approachtermed “peptide threading” can be applied, and in addition a database ofhuman MHC class II binding peptides can be searched for motifs presentin the murine VH and VL sequences, as described in WO 98/52976 and WO00/34317. These motifs bind to any of the 18 major MHC class II DRallotypes, and thus constitute potential T cell epitopes. PotentialT-cell epitopes detected can be eliminated by substituting small numbersof amino acid residues in the variable regions, or preferably, by singleamino acid substitutions. As far as possible conservative substitutionsare made, often but not exclusively, an amino acid common at thisposition in human germline antibody sequences may be used. Humangermline sequences are disclosed in Tomlinson, I. A. et al. J. Mol.Biol. 227:776-798 (1992); Cook, G. P. et al. Immunol. Today Vol. 16 (5):237-242 (1995); Chothia, D. et al. J. Mol. Bio. 227:799-817 (1992). TheV BASE directory provides a comprehensive directory of humanimmunoglobulin variable region sequences (compiled by Tomlinson, I. A.et al. MRC Centre for Protein Engineering, Cambridge, UK). After thedeimmunized VH and VL of an anti-OV064 antibody are constructed bymutagenesis of the murine VH and VL genes, the mutagenized variablesequence can, optionally, be fused to a human constant region, e.g.,human IgG1 or κ constant regions.

Anti-OV064 antibodies that are not intact antibodies are also useful inthis invention. Such antibodies may be derived from any of theantibodies described above. For example, antigen-binding fragments, aswell as full-length monomeric, dimeric or trimeric polypeptides derivedfrom the above-described antibodies are themselves useful. Usefulantibody homologs of this type include (i) a Fab fragment, a monovalentfragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)₂fragment, a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment consisting ofthe VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VHdomains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,Nature 341:544-546 (1989)), which consists of a VH domain; (vii) asingle domain functional heavy chain antibody, which consists of a VHHdomain (known as a nanobody) see e.g., Cortez-Retamozo, et al., CancerRes. 64: 2853-2857 (2004), and references cited therein; and (vii) anisolated complementarity determining region (CDR), e.g., one or moreisolated CDRs together with sufficient framework to provide an antigenbinding fragment. Furthermore, although the two domains of the Fvfragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. Science 242:423-426 (1988); and Huston et al. Proc.Natl. Acad. Sci. USA 85:5879-5883 (1988). Such single chain antibodiesare also intended to be encompassed within the term “antigen-bindingfragment” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies. Antibody fragments, such as Fv, F(ab′)₂ and Fab may beprepared by cleavage of the intact protein, e.g. by protease or chemicalcleavage.

In one approach, consensus sequences encoding the heavy and light chainJ regions may be used to design oligonucleotides for use as primers tointroduce useful restriction sites into the J region for subsequentlinkage of V region segments to human C region segments. C region cDNAcan be modified by site directed mutagenesis to place a restriction siteat the analogous position in the human sequence.

Expression vectors include plasmids, retroviruses, cosmids, YACs, EBVderived episomes, and the like. A convenient vector is one that encodesa functionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, Examples of suitable vectors that can be usedinclude those that are suitable for mammalian hosts and based on viralreplication systems, such as simian virus 40 (SV40), Rous sarcoma virus(RSV), adenovirus 2, bovine papilloma virus (BPV), papovavirus BK mutant(BKV), or mouse and human cytomegalovirus (CMV), and moloney murineleukemia virus (MMLV), native Ig promoters, etc.

Expression in eukaryotic host cells is useful because such cells aremore likely than prokaryotic cells to assemble and secrete a properlyfolded and immunologically active antibody. However, any antibodyproduced that is inactive due to improper folding may be renaturableaccording to well known methods (Kim and Baldwin, “SpecificIntermediates in the Folding Reactions of Small Proteins and theMechanism of Protein Folding”, Ann. Rev. Biochem. 51, pp. 459-89(1982)). It is possible that the host cells will produce portions ofintact antibodies, such as light chain dimers or heavy chain dimers,which also are antibody homologs according to the present invention.

Further, human antibodies or antibodies from other species can begenerated through display-type technologies, including, withoutlimitation, phage display, retroviral display, ribosomal display, andother techniques, using techniques well known in the art and theresulting molecules can be subjected to additional maturation, such asaffinity maturation, as such techniques are well known in the art.Winter and Harris Immunol Today 14:43-46 (1993) and Wright et al. Crit.Reviews in Immunol. 12125-168 (1992). Hanes and Plucthau PNAS USA94:4937-4942 (1997) (ribosomal display), Parmley and Smith Gene73:305-318 (1988) (phage display), Scott TIBS17:241-245 (1992), Cwirlaet al. Proc Natl Acad Sci USA 87:6378-6382 (1990), Russel et al. Nucl.Acids Research 21:1081-1085 (1993), Hoganboom et al. Immunol. Reviews130:43-68 (1992), Chiswell and McCafferty TIBTECH 10:80-84 (1992), andU.S. Pat. No. 5,733,743. If display technologies are utilized to produceantibodies that are not human, such antibodies can be humanized asdescribed above.

It will be appreciated that antibodies that are generated need notinitially possess a particular desired isotype but, rather, the antibodyas generated can possess any isotype and the antibody can be isotypeswitched thereafter using conventional techniques that are well known inthe art. Such techniques include the use of direct recombinanttechniques (see e.g., U.S. Pat. No. 4,816,397), cell-cell fusiontechniques (see e.g., U.S. Pat. No 5,916,771), among others. In thecell-cell fusion technique, a myeloma or other cell line is preparedthat possesses a heavy chain with any desired isotype and anothermyeloma or other cell line is prepared that possesses the light chain.Such cells can, thereafter, be fused and a cell line expressing anintact antibody can be isolated.

By way of example, many of the OV064 antibodies discussed herein arehuman anti-OV064 IgG1 antibodies. Since such antibodies possess desiredbinding to the OV064 molecule, any one of such antibodies can be readilyisotype switched to generate a human IgG4 isotype, for example, whilestill possessing the same variable region (which defines the antibody'sspecificity and affinity, to a certain extent). Accordingly, as antibodycandidates are generated that meet desired “structural” attributes asdiscussed above, they can generally be provided with at least certainadditional “functional” attributes that are desired through isotypeswitching.

Design and Generation of Other Therapeutics

In accordance with the present invention and based on the activity ofthe antibodies that are produced and characterized herein with respectto OV064, the design of other therapeutic modalities including otherantibodies, other antagonists, or chemical moieties other thanantibodies is facilitated. Such modalities include, without limitation,antibodies having similar binding activity or functionality, advancedantibody therapeutics, such as bispecific antibodies, immunoconjugates,and radiolabeled therapeutics, generation of peptide therapeutics,particularly intrabodies, and small molecules. Furthermore, as discussedabove, the effector function of the antibodies of the invention may bechanged by isotype switching to an IgG1, IgG2, IgG3, IgG4, IgD, IgA,IgE, or IgM for various therapeutic uses.

In connection with bispecific antibodies, bispecific antibodies can begenerated that comprise (i) two antibodies one with a specificity toOV064 and another to a second molecule that are conjugated together,(ii) a single antibody that has one chain specific to OV064 and a secondchain specific to a second molecule, or (iii) a single chain antibodythat has specificity to OV064 and the other molecule. Such bispecificantibodies can be generated using techniques that are well known. Forexample, bispecific antibodies may be produced by crosslinking two ormore antibodies (of the same type or of different types). Suitablecrosslinkers include those that are heterobifunctional, having twodistinctly reactive groups separated by an appropriate spacer (e.g.,m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional(e.g., disuccinimidyl suberate). Such linkers are available from PierceChemical Company, Rockford, Ill. See also, e.g., Fanger et al. ImmunolMethods 4:72-81 (1994) and Winter and Harris Immunol Today 14:43-46(1993) and Wright et al. Crit. Reviews in Immunol. 12125-168 (1992), andin connection with (iii) see e.g., Traunecker et al. Int. J. Cancer(Suppl.) 7:51-52 (1992). Songsivilai & Lachmann Clin. Exp. Immunol. 79:315-321 (1990), Kostelny et al. J. Immunol. 148:1547-1553 (1992).

In addition, “Kappabodies” (Ill. et al. “Design and construction of ahybrid immunoglobulin domain with properties of both heavy and lightchain variable regions” Protein Eng 10:949-57 (1997)), “Minibodies”(Martin et al. EMBO J. 13:5303-9 (1994)), “Diabodies” (Holliger et al.Proc Natl Acad Sci USA 90:6444-6448 (1993)), or “Janusins” (Trauneckeret al. EMBO J. 10:3655-3659 (1991) and Traunecker et al. Int J CancerSuppl 7:51-52 (1992)) may also be prepared.

Nucleic Acid and Polypeptides

In another embodiment, the present invention relates to polynucleotideand polypeptide sequences that encode for the antibodies or fragmentsthereof described herein. Such polynucleotides encode for both thevariable and constant regions of each of the heavy and light chains,although other combinations are also contemplated by the presentinvention in accordance with the compositions described herein. Thepresent invention also contemplates oligonucleotide fragments derivedfrom the disclosed polynucleotides and nucleic acid sequencescomplementary to these polynucleotides.

The polynucleotides can be in the form of RNA or DNA. Polynucleotides inthe form of DNA, cDNA, genomic DNA, nucleic acid analogs and syntheticDNA are within the scope of the present invention. The DNA may bedouble-stranded or single-stranded, and if single stranded, may be thecoding (sense) strand or non-coding (anti-sense) strand. The codingsequence that encodes the polypeptide may be identical to the codingsequence provided herein or may be a different coding sequence whichcoding sequence, as a result of the redundancy or degeneracy of thegenetic code, encodes the same polypeptide as the DNA provided herein.

The provided polynucleotides encode at least one heavy chain variableregion and at least one light chain variable region of the presentinvention. Examples of such polynucleotides are shown in SEQ ID NOS: 1,3, 5, 7, 9, and 11 as well as fragments, complements and degeneratecodon equivalents thereof. For example, SEQ ID NO: 1 encodes for theheavy chain of sc77 and SEQ ID NO:3 encodes for the light chain of sc77.SEQ ID NO:5 encodes for the heavy chain of sc189 and SEQ ID NO: 7encodes for the light chain of sc189. SEQ ID NO:9 encodes for the heavychain of sc209 and SEQ ID NO: 11 encodes for the light chain of sc209.

The present invention also includes variant polynucleotides containingmodifications such as polynucleotide deletions, substitutions oradditions, and any polypeptide modification resulting from the variantpolynucleotide sequence. A polynucleotide of the present invention mayalso have a coding sequence that is a variant of the coding sequenceprovided herein.

It is contemplated that polynucleotides will be considered to hybridizeto the sequences provided herein if there is at least 50%, 60%, 70%,75%, 80%, 85%, 90%, 95% identity between the polynucleotide and thesequence.

The present invention further relates to polypeptides that encode forthe antibodies of the present invention as well as fragments, analogsand derivatives of such polypeptides. The polypeptides of the presentinvention may be recombinant polypeptides, naturally producedpolypeptides or synthetic polypeptides. The fragment, derivative oranalogs of the polypeptides of the present invention may be one in whichone or more of the amino acid residues is substituted with a conservedor non-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code; or it may be one in which one or more ofthe amino acid residues includes a substituent group; or it may be onein which the polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol); or it may be one in which the additional aminoacids are fused to the polypeptide, such as a leader or secretorysequence or a sequence that is employed for purification of thepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are within the scope of the present invention. In variousaspects, the polypeptides of the invention may be partially purified, orpurified product.

A polypeptide of the present invention may have an amino acid sequencethat is identical to that of the antibodies described herein or that isdifferent by minor variations due to one or more amino acidsubstitutions. The variation may be a “conservative change” typically inthe range of about 1 to 5 amino acids, wherein the substituted aminoacid has similar structural or chemical properties, e.g., replacement ofleucine with isoleucine or threonine with serine; replacement of lysinewith arginine or histidine. In contrast, variations may includenonconservative changes, e.g., replacement of a glycine with atryptophan. Similar minor variations may also include amino aciddeletions or insertions or both. Guidance in determining which and howmany amino acid residues may be substituted, inserted, or deletedwithout changing biological or immunological activity may be found usingcomputer programs well known in the art, for example DNASTAR software(DNASTAR, Inc., Madison, Wis.).

The provided polypeptides encode at least one heavy chain variableregion or at least one light chain variable region of the antibodies ofthe present invention. The provided polypeptides can encode at least oneheavy chain variable region and one light chain variable region of theantibodies of the present invention. Examples of such polypeptides arethose having the amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8,10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, and 30, and fragments thereof. Specifically, the heavy chain of sc77has the amino acid sequence shown in SEQ ID NO: 2 and the light chainhas the amino acid sequence shown in SEQ ID NO:4. The amino acidsequence of the heavy chain of sc 189 is shown in SEQ ID NO:6 and thelight chain has the amino acid sequence shown in SEQ ID NO:8. The aminoacid sequence of the heavy chain of sc209 is shown in SEQ ID NO:10 andthe light chain has the amino acid sequence shown in SEQ ID NO:12. Theheavy chain CDR sequences of sc77 have the amino acid sequence shown inSEQ ID NO: 13 (HCDR1), SEQ ID NO:14 (HCDR2) and SEQ ID NO:15 (HCDR3);and the light chain CDRs have the amino acid sequence shown in SEQ IDNO:22 (LCDR1); SEQ ID NO:23 (LCDR2); and SEQ ID NO:24 (LCDR3). The aminoacid sequence of the heavy chain CDRs of sc189 are shown in SEQ ID NO:16(HCDR1), SEQ ID NO:17 (HCDR2), and SEQ ID NO:18 (HCDR3); and the lightchain CDRs have the amino acid sequences shown in SEQ ID NO:25 (LCDR1),SEQ ID NO:26 (LCDR2), and SEQ ID NO:27 (LCDR3). The amino acid sequenceof the heavy chain CDRs of sc209 are shown in SEQ ID NO:19 (HCDR1), SEQID NO:20 (HCDR2), and SEQ ID NO:21 (HCDR3) and the light chain CDRs havethe amino acid sequences shown in SEQ ID NO:28 (LCDR1), SEQ ID NO:29(LCDR2) and SEQ ID NO:30 (LCDR3).

The present invention also provides vectors that include thepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the present invention and theproduction of the antibodies of the present invention by recombinanttechniques.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted intoappropriate restriction endonuclease sites by procedures known in theart. The polynucleotide sequence in the expression vector is operativelylinked to an appropriate expression control sequence (i.e. promoter) todirect mRNA synthesis. Examples of such promoters include, but are notlimited to, the LTR or the SV40 promoter, the E. coli lac or trp, thephage lambda P_(L) promoter and other promoters known to controlexpression of genes in prokaryotic or eukaryotic cells or their viruses.The expression vector also contains a ribosome binding site fortranslation initiation and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression. Forexample, the vector can contain enhancers, which aretranscription-stimulating DNA sequences of viral origin, such as thosederived form simian virus such as SV40, polyoma virus, cytomegalovirus,bovine papilloma virus or Moloney sarcoma virus, or genomic, origin. Thevector preferably also contains an origin of replication. The vector canbe constructed to contain an exogenous origin of replication or, such anorigin of replication can be derived from SV40 or another viral source,or by the host cell chromosomal replication mechanism.

In addition, the vectors optionally contain a marker gene for selectionof transfected host cells such as dihydrofolate reductase orantibiotics, such as neomycin, GA418 (geneticin, a neomycin-derivative)or hygromycin, or genes which complement a genetic lesion of the hostcells such as the absence of thymidine kinase, hypoxanthinephosphoribosyl transferase, dihydrofolate reductase, etc.

In order to obtain the antibodies of the present invention, one or morepolynucleotide sequences that encode for the light and heavy chainvariable regions and light and heavy chain constant regions of theantibodies of the present invention should be incorporated into avector. Polynucleotide sequences encoding the light and heavy chains ofthe antibodies of the present invention can be incorporated into one ormultiple vectors and then incorporated into the host cells.

As will be appreciated, antibodies in accordance with the presentinvention can be expressed in cell lines other than hybridoma celllines. Sequences encoding the cDNAs or genomic clones for the particularantibodies can be used for a suitable mammalian or nonmammalian hostcells. Transformation can be by any known method for introducingpolynucleotides into a host cell, including, for example packaging thepolynucleotide in a virus (or into a viral vector) and transducing ahost cell with the virus (or vector) or by transfection procedures knownin the art, for introducing heterologous polynucleotides into mammaliancells, e.g., dextran-mediated transfection, calcium phosphateprecipitation, polybrene mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) into liposomesand direct microinjection of the DNA molecule. The transformationprocedure used depends upon the host to be transformed. Methods forintroduction of heterologous polynucleotides into mammalian cells arewell known in the art and include, but are not limited to,dextran-mediated transfection, calcium phosphate precipitation,polybrene mediated transfection, protoplast fusion, electroporation,particle bombardment, encapsulation of the polynucleotide(s) inliposomes, peptide conjugates, dendrimers, and direct microinjection ofthe DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC), including but not limited toChinese hamster ovary (CHO) cells, NSO cells, HeLa cells, baby hamsterkidney (BHK) cells, monkey kidney cells (COS), human hepatocellularcarcinoma cells (e.g., Hep G2), and a number of other cell lines.Non-mammalian cells including but not limited to bacterial, yeast,insect, and plants can also be used to express recombinant antibodies.Site directed mutagenesis of the antibody CH2 domain to eliminateglycosylation may be preferred in order to prevent changes in either theimmunogenicity, pharmacokinetic, and/or effector functions resultingfrom non-human glycosylation. The expression methods are selected bydetermining which system generates the highest expression levels andproduce antibodies with constitutive OV064 binding properties.

Further, expression of antibodies of the invention (or other moietiestherefrom) from production cell lines can be enhanced using a number ofknown techniques. For example, the glutamine sythetase and DHFR geneexpression systems are common approaches for enhancing expression undercertain conditions. High expressing cell clones can be identified usingconventional techniques, such as limited dilution cloning, Microdroptechnology, or any other methods known in the art. The GS system isdiscussed in whole or part in connection with European Patent Nos. 0 216846, 0 256 055, and 0 323 997 and European Patent Application No.89303964.4.

In an exemplary system for recombinant expression of a modifiedantibody, or antigen-binding portion thereof, of the invention, arecombinant expression vector encoding both the antibody heavy chain andthe antibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to enhancer/promoter regulatory elements (e.g., derived fromSV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLPpromoter regulatory element or an SV40 enhancer/AdMLP promoterregulatory element) to drive high levels of transcription of the genes.The recombinant expression vector also carries a DHFR gene, which allowsfor selection of CHO cells that have been transfected with the vectorusing methotrexate selection/amplification. The selected transformanthost cells are cultured to allow for expression of the antibody heavyand light chains and intact antibody is recovered from the culturemedium. Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the antibody from theculture medium.

Antibodies of the invention can also be produced transgenically throughthe generation of a mammal or plant that is transgenic for theimmunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies can beproduced in, and recovered from, the milk of goats, cows, or othermammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and5,741,957.

Antibodies in accordance with the present invention have been analyzedstructurally and functionally. In connection with the structures of theantibodies, amino acid sequences of each of the human heavy and kappalight chains were predicted based on cDNA sequences obtained throughRT-PCR of the hybridomas. See Examples, Table 1, Table 2, SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12. N-terminal sequencing of the antibodies was alsoconducted in confirmation of the results discussed in Examples toconfirm sequences of the human antibody chains. Kinetic analyses of theantibodies were conducted to determine affinities. See Examples, Table4. Antibodies in accordance with the invention have high affinities forOV064. Additionally, binding characteristics of the antibodies wasconducted to determine epitopes which the antibodies recognize. Further,antibodies were analyzed for internalization into cells expressingOV064, by reducing gel electrophoresis (SDS-PAGE), western blotanalysis, and antibody production by the hybridomas or host cellsproducing antibodies was assessed. See Examples and Tables below.

Conjugates

Several properties of Ov064 make it a suitable target forimmunoconjugate tumor activated prodrug (TAP) or immunotoxin conjugatedevelopment: first, it is highly expressed on a large fraction of mostovarian cancers, and significantly expressed in lung and breast cancers;second, Ov064 rapidly internalizes and is capable of bringing cytotoxicconcentrations of toxin (e.g., maytansine, e.g., DM1, DM4) into cells;third, Ov064 is expressed at very low levels or is absent in most normalhuman tissues and finally, Ov064 shows evidence of being polarized innormal tissue that express the antigen. The invention thus providesmonoclonal antibodies which are internalized and capable of deliveringtoxic payloads of immunoconjugate into cells expressing OV064, but notinto cells where the target is not expressed. The toxic payload mayinclude immunotoxin comprising a cytotoxic agent Immunoconjugates ofantibodies, e.g., modified monoclonal antibodies and fully human mAbantibodies, and antigen binding fragments or derivatives thereof whichspecifically bind the extracellular domain of OV064, are provided.

As used herein, “immunoconjugate” comprises an anti-OV064 antibody orantigen binding fragment or derivative thereof, which is conjugated toanother entity (e.g., to a cytotoxic or cytostatic moiety, a labelmoiety, a therapeutic moiety) or modified as described in more detailherein. As used herein, a “moiety” of an immunoconjugate is intended torefer to a component of the conjugate (e.g., an immunoglobulin moiety(i.e., an antibody or antigen binding fragment or derivative thereof), atherapeutic moiety, a cytotoxic moiety). For example, a cytotoxic moietyof an immunoconjugate comprises a cytotoxic agent which is conjugated toan antibody or antigen binding fragment. An anti-OV064 antibody or anantigen binding fragment thereof may be conjugated to another molecularentity, e.g., a cytotoxic or cytostatic agent, e.g., a therapeuticagent, a drug, a compound emitting radiation, molecules of plant,fungal, or bacterial origin, or a biological protein (e.g., a proteintoxin) or particle (e.g., a recombinant viral particle, e.g., via aviral coat protein); a detectable agent; a pharmaceutical agent; and/ora protein or peptide that can mediate association of the antibody orantibody portion with another molecule (such as a streptavidin coreregion or a polyhistidine tag). For example, an antibody or antibodyportion of the invention can be functionally linked by any suitablemethod (e.g., chemical coupling, genetic fusion, noncovalent associationor otherwise) to one or more other molecular entities. Examples oflinkers capable of being used to couple an immunotoxin to an antibody orantibody portion of the invention include, for example, N-succinimidyl3-(2-pyridyldithio)proprionate (also known as N-succinimidyl4-(2-pyridyldithio)pentanoate or SPP);4-succinimidyl-oxycarbonyl-a-(2-pyridyldithio)-toluene (SMPT);N-succinimidyl-3-(2-pyridyldithio)butyrate (SDPB); 2-iminothiolane;S-acetylsuccinic anhydride; disulfide benzyl carbamate; carbonate;hydrazone linkers; N-(α-Maleimidoacetoxy) succinimide ester;N-[4-(p-Azidosalicylamido)butyl]-3″-(2″-pyridyldithio)propionamide(AMAS); N-[β-Maleimidopropyloxy]succinimide ester (BMPS);[N-e-Maleimidocaproyloxy]succinimide ester (EMCS);N-[g-Maleimidobutyryloxy]succinimide ester (GMBS);Succinimidyl-4-[N-Maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate](LC-SMCC); Succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate(LC-SPDP); m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS);N-Succinimidyl[4-iodoacetyl]aminobenzoate (SIAB); Succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC); N-Succinimidyl3-[2-pyridyldithio]propionamido (SPDP);[N-e-Maleimidocaproyloxy]sulfosuccinimide ester (Sulfo-EMCS);N-[g-Maleimidobutyryloxy]sulfosuccinimide ester (Sulfo-GMBS);4-Sulfosuccinimidyl-6-methyl-α-(2-pyridyldithio)toluamido]hexanoate)(Sulfo-LC-SMPT); Sulfosuccinimidyl6-(3′-[2-pyridyldithio]-propionamido)hexanoate (Sulfo-LC-SPDP);m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Sulfo-MBS);N-Sulfosuccinimidyl[4-iodoacetyl]aminobenzoate (Sulfo-SIAB);Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate(Sulfo-SMCC); Sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate(Sulfo-SMPB); EGS; DST; DOTA; DTPA; and thiourea linkers.

In connection with immunoconjugates, the provided antibodies or antigenbinding fragments thereof can be modified to act as immunoconjugatesutilizing techniques that are well known in the art. See e.g., VitettaImmunol Today 14:252 (1993). See also U.S. Pat. No. 5,194,594. Inconnection with the preparation of radiolabeled antibodies, suchmodified antibodies can also be readily prepared utilizing techniquesthat are well known in the art. See e.g., Junghans et al. in CancerChemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo,eds., Lippincott Raven (1996)). See also U.S. Pat. Nos. 4,681,581,4,735,210, 5,101,827, 5,102,990 (U.S. Re. Pat. No. 35,500), 5,648,471,and 5,697,902. Immunoconjugates comprising toxins, tumor activatedprodrugs, and radiolabeled molecules would be likely to kill cellsexpressing OV064, and particularly in those cells in which the providedantibodies and antigen binding fragments of the invention are effective(e.g., tumor cells expressing OV064, e.g., ovarian, lung, breast tumorcells).

As discussed, the antibody, antigen binding fragment, or derivativethereof can be conjugated to a therapeutic agent. For example, theanti-OV064 antibody, or antigen-binding fragment thereof, can be coupledto a biological protein, a molecule of plant or bacterial origin (orderivative thereof), e.g., a maytansinoid (e.g., maytansinol, e.g., DM1,DM4), a taxane, a calicheamicin, a duocarmycin, or derivatives thereof.The maytansinoid can be, for example, maytansinol or a maytansinolanalogue. Examples of maytansinol analogues include those having amodified aromatic ring (e.g., C-19-decloro, C-20-demethoxy,C-20-acyloxy) and those having modifications at other positions (e.g.,C-9-CH, C-14-alkoxymethyl, C-14-hydroxymethyl or aceloxymethyl,C-15-hydroxy/acyloxy, C-15-methoxy, C-18-N-demethyl, 4,5-deoxy).Maytansinol and maytansinol analogues are described, for example, inU.S. Pat. Nos. 5,208,020, 6,333,410, the contents of which isincorporated herein by reference. Maytansinol can be coupled toantibodies using, e.g., an N-succinimidyl 3-(2-pyridyldithio)proprionate(also known as N-succinimidyl 4-(2-pyridyldithio)pentanoate or SPP),4-succinimidyl-oxycarbonyl-a-(2-pyridyldithio)-toluene (SMPT),N-succinimidyl-3-(2-pyridyldithio)butyrate (SDPB), 2-iminothiolane, orS-acetylsuccinic anhydride. The taxane can be, for example, a taxol,taxotere, or novel taxane (see, e.g., International Patent PublicationNo. WO 01/38318, published May 31, 2001). The calicheamicin can be, forexample, a bromo-complex calicheamicin (e.g., an alpha, beta or gammabromo-complex), an iodo-complex calicheamicin (e.g., an alpha, beta orgamma iodo-complex), or analogs and mimics thereof. Bromo-complexcalicheamicins include I₁—BR, I₂—BR, I₃—BR, I₄—BR, Θ₁-BR, Θ₂-BR andK₁—BR. Iodo-complex calicheamicins include I₁—I, I₂—I, I₃—I, Θ₁-I, Θ₂-I,Λ₁-I and K₁—BR. Calicheamicin and mutants, analogs and mimics thereofare described, for example, in U.S. Pat. Nos. 4,970,198, issued Nov. 13,1990, 5,264,586, issued Nov. 23, 1993, 5,550,246, issued Aug. 27, 1996,5,712,374, issued Jan. 27, 1998, and 5,714,586, issued Feb. 3, 1998, thecontents of which are incorporated herein by reference. Duocarmycinanalogs (e.g., KW-2189, DC88, DC89 CBI-TMI, and derivatives thereof aredescribed, for example, in U.S. Pat. No. 5,070,092, U.S. Pat. No.5,187,186, U.S. Pat. No. 5,641,780, U.S. Pat. No. 5,641,780, U.S. Pat.No. 4,923,990, and U.S. Pat. No. 5,101,038, the contents of which areincorporated herein by reference.

Examples of other therapeutic agents include, but are not limited to,cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide,tenoposide, colchicin, dihydroxy anthracin dione, mitoxantrone,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065 (see U.S.Pat. Nos. 5,475,092, 5,585,499, 5,846,545), melphalan, carmustine (BSNU)and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, mitomycin, puromycin anthramycin (AMC)),duocarmycin and analogs or derivatives thereof, and anti-mitotic agents(e.g., vincristine, vinblastine, taxol, auristatins (e.g., auristatin E)and maytansinoids, and analogs or homologs thereof.

The conjugates of the invention can be used for modifying a givenbiological response. The therapuetic agent is not to be construed aslimited to classical chemical therapeutic agents. For example, thetherapeutic agent may be a protein or polypeptide possessing a desiredbiological activity. Such proteins may include, for example, a toxinsuch as abrin, ricin A, pseudomonas exotoxin, gelonin, diphtheria toxin,or a component thereof (e.g., a component of pseudomonas exotoxin isPE38); a protein such as tumor necrosis factor, interferon, nerve growthfactor, platelet derived growth factor, tissue plasminogen activator;or, biological response modifiers such as, for example, lymphokines,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or other growth factors. Similarly,the therapeutic agent can be a viral particle, e.g., a recombinant viralparticle, that is conjugated (e.g., via a chemical linker) or fused(e.g., via a viral coat protein) to an anti-OV064 antibody of theinvention. Introduction of the viral nucleic acid molecules, e.g.,recombinant viral nucleic acid molecules, into cells, e.g., cancercells, e.g., ovarian, breast or lung cancer cells associated with tumorsthat express OV064 can occur following binding and endocytosis of theanti-OV064 antibody/viral particle conjugate or fusion.

Therapeutically active radioisotopes can also be coupled to anti-OV064antibodies, or antigen binding fragments, or derivatives thereof.Radioactive isotopes can be used in diagnostic or therapeuticapplications. Radioactive isotopes that can be coupled to the anti-OV064antibodies include, but are not limited to a-, b-, or g-emitters, or b-and g-emitters. Such radioactive isotopes include, but are not limitedto iodine (¹³¹I or ¹²⁵I), yttrium (⁹⁰Y), lutetium (¹⁷⁷Lu), actinium(²²⁵Ac), praseodymium, astatine (²¹¹At), rhenium (¹⁸⁶Re), bismuth (²¹²Bior ²¹³Bi), indium (¹¹¹In), technetium (⁹⁹mTc), phosphorus (³²P), rhodium(¹⁸⁸Rh), sulfur (³⁵S), carbon (¹⁴C), tritium (³H), chromium (⁵¹Cr),chlorine (³⁶Cl), cobalt (⁵⁷Co or ⁵⁸Co), iron (⁵⁹Fe), selenium (⁷⁵Se), orgallium (⁶⁷Ga). Radioisotopes useful as therapeutic agents includeyttrium (⁹⁰Y), lutetium (¹⁷⁷Lu), actinium (²²⁵Ac), praseodymium,astatine (²¹¹At), rhenium (¹⁸⁶Re), bismuth (²¹²Bi or ²¹³Bi), and rhodium(¹⁸⁸Rh). Radioisotopes useful as labels, e.g., for use in diagnostics,include iodine (¹³¹I or ¹²⁸I), indium (¹¹¹In), technetium (⁹⁹mTc),phosphorus (³²P), carbon (¹⁴C), and tritium (³H), or one or more of thetherapeutic isotopes listed above.

Useful detectable agents with which an antibody or an antibody portionof the invention may be derivatized (or labeled) to include fluorescentcompounds, various enzymes, prosthetic groups, luminescent materials,bioluminescent materials, fluorescent emitting metal atoms, e.g.,europium (Eu), and other anthanides, and radioactive materials(described above). Exemplary fluorescent detectable agents includefluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and thelike. An antibody may also be derivatized with detectable enzymes, suchas alkaline phosphatase, horseradish peroxidase, b-galactosidase,acetylcholinesterase, glucose oxidase and the like. When an antibody isderivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with a prosthetic group(e.g., streptavidin/biotin and avidin/biotin). For example, an antibodymay be derivatized with biotin, and detected through indirectmeasurement of avidin or streptavidin binding. Examples of suitablefluorescent materials, include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of bioluminescent materials include luciferase,luciferin, and aequorin.

Pharmaceutical Compositions

In another aspect, the present invention provides compositions, e.g.,pharmaceutically acceptable compositions, which include an antibody oran antigen binding fragment molecule, or derivative thereof describedherein, formulated together with a pharmaceutically acceptable carrier.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, isotonic and absorption delaying agents,and the like that are physiologically compatible. The carrier can besuitable for intravenous, intramuscular, subcutaneous, parenteral,rectal, spinal or epidermal administration (e.g., by injection orinfusion).

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, liposomes and suppositories. The preferred form dependson the intended mode of administration and therapeutic application.Typical compositions are in the form of injectable or infusiblesolutions. The mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In some embodiments, theantibody is administered by intravenous infusion or injection. In otherembodiments, the antibody is administered by intramuscular orsubcutaneous injection.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high antibody concentration. Sterileinjectable solutions can be prepared by incorporating the activecompound (i.e., antibody or antibody portion) in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, theprovided methods of preparation are vacuum drying and freeze-drying thatyields a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theproper fluidity of a solution can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

The antibodies and antigen binding fragments of the present inventioncan be administered by a variety of methods known in the art, althoughfor many therapeutic applications, the route/mode of administration isintravenous injection or infusion. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. In certain embodiments, the active compoundmay be prepared with a carrier that will protect the compound againstrapid release, such as a controlled release formulation, includingimplants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, an antibody or an antibody portion of theinvention may be orally administered, for example, with an inert diluentor an assimilable edible carrier. The compound (and other ingredients ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, buccal tablets, troches, capsules, elixiers,suspensions, syrups, wafers, and the like. To administer an antibody oran antibody fragment of the invention by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.

Therapeutic compositions can be administered with medical devices knownin the art.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or an antigen bindingfragment of the invention is 0.1-20 mg/kg, or 1-10 mg/kg. It is to benoted that dosage values may vary with the type and severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

The pharmaceutical compositions of the invention may include a“therapeutically effective” amount of an antibody or an antigen bindingfragment of the invention. A “therapeutically effective” amount refersto an amount effective, at dosages and for periods of time necessary, toachieve the desired therapeutic result. A therapeutically effectiveamount of the modified antibody or antibody fragment may vary accordingto factors such as the disease state, age, sex, and weight of theindividual, and the ability of the antibody or antibody portion toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of themodified antibody or antibody fragment is outweighed by thetherapeutically beneficial effects. A “therapeutically effective dosage”preferably inhibits a measurable parameter, e.g., tumor growth rate byat least about 20%, at least about 40%, at least about 60%, and in someaspects preferably at least about 80% relative to untreated subjects.The ability of a compound to inhibit a measurable parameter, e.g.,cancer, can be evaluated in an animal model system predictive ofefficacy in human tumors. Alternatively, this property of a compositioncan be evaluated by examining the ability of the compound to inhibit,such inhibition in vitro by assays known to the skilled practitioner.

Also within the scope of the invention are kits comprising an anti-OV064antibody, or antigen-binding fragment thereof. Also included in theinvention are kits comprising immunoconjugates comprising an anti-OV064antibody or antigen binding fragment conjugated to a cytotoxic moiety ora detectable label. Further included are kits comprising liposomecompositions comprising anti-OV064 antibodies or antigen bindingfragments thereof. The kit can include one or more other elementsincluding: instructions for use; other reagents, e.g., a label, atherapeutic agent, or an agent useful for chelating, or otherwisecoupling, an antibody to a label or therapeutic agent, or aradioprotective composition; devices or other materials for preparingthe antibody for administration; pharmaceutically acceptable carriers;and devices or other materials for administration to a subject.Instructions for use can include instructions for diagnosticapplications of the anti-OV064 antibodies (or antigen-binding fragmentthereof) to detect OV064, in vitro, e.g., in a sample, e.g., a biopsy orcells from a patient having a cancer, or in vivo. The instructions caninclude instructions for therapeutic application including suggesteddosages and/or modes of administration, e.g., in a patient with a cancer(e.g., ovarian, breast, lung cancer). Other instructions can includeinstructions on coupling of the antibody to a chelator, a label or atherapeutic agent, or for purification of a conjugated antibody, e.g.,from unreacted conjugation components. As discussed above, the kit caninclude a label, e.g., any of the labels described herein. As discussedabove, the kit can include a therapeutic agent, e.g., a therapeuticagent described herein. In some applications the antibody will bereacted with other components, e.g., a chelator or a label ortherapeutic agent, e.g., a radioisotope, e.g., yttrium or lutetium. Insuch cases the kit can include one or more of a reaction vessel to carryout the reaction or a separation device, e.g., a chromatographic column,for use in separating the finished product from starting materials orreaction intermediates.

The kit can further contain at least one additional reagent, such as adiagnostic or therapeutic agent, e.g., a diagnostic or therapeutic agentas described herein, and/or one or more additional anti-OV064 antibodies(or fragments thereof), formulated as appropriate, in one or moreseparate pharmaceutical preparations.

The kit can further contain a radioprotectant. The radiolytic nature ofisotopes, e.g., ⁹⁰Yttrium (⁹⁰Y) is known. In order to overcome thisradiolysis, radioprotectants may be included, e.g., in the reactionbuffer, as long as such radioprotectants are benign, meaning that theydo not inhibit or otherwise adversely affect the labeling reaction,e.g., of an isotope, such as of ⁹⁰Y, to the antibody. The formulationbuffer of the present invention may include a radioprotectant such ashuman serum albumin (HSA) or ascorbate, which minimize radiolysis due toyttrium or other strong radionuclides. Other radioprotectants are knownin the art and can also be used in the formulation buffer of the presentinvention, i.e., free radical scavengers (phenol, suffites, glutathione,cysteine, gentisic acid, nicotinic acid, ascorbyl palmitate, HOP(:O)H₂Iglycerol, sodium formaldehyde sulfoxylate, Na₂S₂0, Na₂S₂0₃, and S0₂,etc.).

A provided kit is one useful for radiolabeling a chelator-conjugatedprotein or peptide with a therapeutic radioisotope for administration toa patient. The kit includes (i) a vial containing chelator-conjugatedantibody, (ii) a vial containing formulation buffer for stabilizing andadministering the radiolabeled antibody to a patient, and (iii)instructions for performing the radiolabeling procedure. The kitprovides for exposing a chelator-conjugated antibody to the radioisotopeor a salt thereof for a sufficient amount of time under amiableconditions, e.g., as recommended in the instructions. A radiolabeledantibody having sufficient purity, specific activity and bindingspecificity is produced. The radiolabeled antibody may be diluted to anappropriate concentration, e.g., in formulation buffer, and administereddirectly to the patient with or without further purification. Thechelator-conjugated antibody may be supplied in lyophilized form.

Uses of the Invention

The antibodies have in vitro and in vivo diagnostic, therapeutic andprophylactic utilities. For example, these antibodies can beadministered to cells in culture, e.g. in vitro or ex vivo, or in asubject, e.g., in vivo, to treat, prevent, and/or diagnose a variety ofdisorders, such as cancers (e.g., ovarian, breast, lung). Morespecifically, antibodies or antibody fragments of the invention andcompositions comprising the provided antibodies or antibody fragment(e.g., immunoconjugates (e.g., coupled to cytotoxic agents), can be usedto treat cancers (e.g., a cancer where the target protein is expressedin at least some of the cells, e.g., ovarian cancer, breast cancer, lungcancer). The antibodies or antigen binding fragments of the invention,and immunoconjugates or compositions comprising the antibodies orantigen binding fragments provided herein can be used to treat cancers(e.g., ovarian cancer, breast cancer, lung cancer) in which OV064 isexpressed. The provided antibody or antigen binding fragments canfurthermore be used in cancer immunotherapy by blocking tumor inducedimmune silencing to inhibit T-cell proliferation and activation.Furthermore, the anti-OV064 antibodies or antigen binding fragmentsprovided and compositions comprising same can also be used to diagnoseand determine what cancers to be treated.

As used herein, the term “subject” is intended to include human andnon-human animals. For example, a subject includes a patient (e.g., ahuman patient, a veterinary patient), having a disorder characterized byan OV064-expressing cell (e.g., a cancer cell). The term “non-humananimals” of the invention includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dog, cow, chickens,amphibians, reptiles, etc.

In one embodiment, the subject is a human subject. Alternatively, thesubject can be a mammal expressing an OV064 antigen with which aantibody of the invention cross-reacts. An antibody or an antigenbinding fragment molecule of the invention can be administered to ahuman subject for therapeutic purposes. Moreover, an anti-OV064antibody, or fragment thereof can be administered to a non-human mammalexpressing the OV064-like antigen with which the antibody cross-reacts(e.g., a primate, pig or mouse) for veterinary purposes or as an animalmodel of human disease. Animal models may be useful for evaluating thetherapeutic efficacy of antibodies of the invention (e.g., testing ofdosages and time courses of administration).

In one embodiment, the invention provides a method of inhibitingOV064-mediated cell signaling or a method of killing a cell. The methodmay be used with any cell or tissue which expresses OV064 such as acancerous cell, (e.g., an ovarian cancer, breast cancer, pancreaticcancer, lung cancer cell), or a metastatic lesion (e.g., a cell found inrenal, urothelial (e.g., bladder), testicular, colon, rectal, lung(e.g., non-small cell lung carcinoma), breast, liver, neural (e.g.,neuroendocrine), glial (e.g., glioblastoma), pancreatic (e.g.,pancreatic duct) cancer and/or metastasic melanoma (e.g., malignantmelanoma), or soft tissue sarcoma). Methods of the invention include thesteps of contacting the cell, with a anti-OV064 antibody or antigenbinding fragment as described herein, or an immunoconjugate orcomposition comprising an anti-OV064 antibody or antigen bindingfragment thereof, in an amount sufficient to inhibit OV064-mediated cellsignaling or an amount sufficient to kill the cell. In methods ofkilling a cell, the method comprises using an immunoconjugate comprisingan anti-OV064 antibody or antigen binding fragment thereof and acytotoxic moiety.

The subject method can be used on cells in culture, e.g. in vitro or exvivo. For example, cells which express OV064 such as cancer cells (e.g.,malignant ovarian, breast, pancreatic or lung cancer cells), metastaticcells (e.g., renal, an urothelial, colon, rectal, lung, ovarian, breast,lung, pancreas, endometrial, or liver, cancerous or metastatic cells),or recombinant cells which express OV064, can be cultured in vitro inculture medium and the contacting step can be effected by adding theanti-OV064 antibody or fragment thereof, to the culture medium. Themethod can be performed on cells present in a subject, as part of an invivo (e.g., therapeutic) protocol. For in vivo embodiments, thecontacting step is effected in a subject and includes administering animmunoconjugate comprising an anti-OV064 antibody or fragment thereofand a cytotoxic moiety to the subject under conditions effective topermit both binding of the antibody or fragment to the extracellulardomain of OV064 expressed on the cell, and the treating of the cell.

Examples of disorders that can be treated include, but are not limitedto, ovarian cancer, breast cancer, lung cancer, pancreatic cancer,endometrial cancer, or metastases thereof, or any cancerous disorderwhich includes at least some expressing OV064 antigen. As used herein,the term “cancer” is meant to include all types of cancerous growths oroncogenic processes, metastatic tissues or malignantly transformedcells, tissues, or organs, irrespective of histopathologic type or stageof invasiveness. The terms cancer or tumor, may be used interchangeably(e.g., used in the context of “treatment of a cancer” or “treatment of atumor”).

Examples of cancerous disorders include, but are not limited to, solidtumors, soft tissue tumors, and metastatic lesions. Examples of solidtumors include malignancies, e.g., sarcomas, adenocarcinomas, andcarcinomas, of the various organ systems, such as those affectingovarian, lung, pancreas, lymphoid, and genitourinary tract (e.g.,endometrial). Adenocarcinomas include malignancies such as non-smallcell carcinoma of the lung. Metastatic lesions of the aforementionedcancers can also be treated or prevented using the methods andcompositions of the invention. The subject method can be useful intreating malignancies of the various organ systems, such as thoseaffecting those previously described. Additionally, the subject methodcan be useful in treating a relevant disorder at any stage orsubclassification. For example, the subject method can be useful in anyof stage 0 breast cancer patients, stage I breast cancer patients, stageIIA breast cancer patients, stage JIB breast cancer patients, stage IIIAbreast cancer patients, stage IIIB breast cancer patients, stage IVbreast cancer patients, grade I breast cancer patients, grade II breastcancer patients, and grade III breast cancer patients, as well as inmalignant breast cancer patients, ductal carcinoma breast cancerpatients, and lobular carcinoma breast cancer patients.

Methods of administering antibody molecules are described above.Suitable dosages of the molecules used will depend on the age and weightof the subject and the particular compound used. The antibody moleculescan be used as competitive agents for ligand binding to inhibit, reducean undesirable interaction (e.g., reduce immunosuppressive effects ofOV064 mediated signaling).

In one embodiment, the anti-OV064 antibodies, or antigen-bindingfragments thereof, can be used to kill or suppress cancerous cells invivo. For example, the anti-OV064 antibodies can be used to treat adisorder described herein. The antibodies, or fragments thereof can beused by themselves or conjugated to a second agent, e.g., a cytotoxicagent, radioisotope, a pro-drug, or a protein, e.g., a protein toxin ora viral protein. This method includes: administering the antibody, aloneor an immunoconjugate where the antibody or antigen binding fragment isconjugated to a cytotoxic agent, to a subject requiring such treatment.

The antibodies of the present invention may be used to deliver a varietyof agents, e.g., a therapeutic agent, a drug, a radioisotope, moleculesof plant, fungal, or bacterial origin, biological proteins (e.g.,protein toxins) or particles (e.g., a recombinant viral particles, e.g.,via a viral coat protein), or mixtures thereof. A therapeutic agent canbe an intracellularly active drug or other agent, such as short-rangeradiation emitters, including, for example, short-range, high-energyα-emitters, as described herein. In some embodiments, the anti-OV064antibody, or antigen binding fragment thereof, can be coupled to amolecule of plant or bacterial origin (or derivative thereof), e.g., amaytansinoid (e.g., maytansinol, e.g., DM1 or DM4 maytansinoid). DM1 isa sulfhydryl-containing derivative of maytansine that can be linked toantibodies via a disulfide linker that releases DM1 when inside targetcells. The disulfide linkers display greater stability in storage and inserum than other linkers. Maytansine is a cytotoxic agent that effectscell killing by preventing the formation of microtubules anddepolymerization of extant microtubules. It is 100- to 1000-fold morecytotoxic than anticancer agents such as doxorubicin, methotrexate, andvinca alkyloid, which are currently in clinical use. In other examples,the anti-OV064 antibody, or antigen binding fragment thereof, can becoupled to a taxane, an auristatin, a duocarmycin, a calicheamicin, or aderivative thereof. Additionally, anti-OV064 antibody or antigen bindingfragment thereof can be coupled to a proteosome inhibitor or atopoisomerase inhibitor. For example,[(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(3-mercaptoacetyl)amino]propyl]amino]butyl]Boronicacid is a suitable proteosome inhibitor, andN,N′-bis[2-(9-methylphenazine-1-carboxamido)ethyl]-1,2-ethanediamine isa suitable topoisomerase inhibitor.

Enzymatically active toxins and fragments thereof are exemplified bydiphtheria toxin A fragment, nonbinding active fragments of diphtheriatoxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin Achain, modeccin A chain, α-sacrin, certain Aleurites fordii proteins,certain Dianthin proteins, Phytolacca americana proteins (PAP, PAPII andPAP-S), Morodica charantia inhibitor, curcin, crotin, Saponariaofficinalis inhibitor, gelonin, mitogillin, restrictocin, phenomycin,and enomycin. In a provided embodiment, the anti-OV064 antibody isconjugated to maytansinoids, e.g., maytansinol (see U.S. Pat. No.5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499,5,846,545). Procedures for preparing enzymatically active polypeptidesof the immunotoxins are described in WO84/03508 and WO85/03508, whichare hereby incorporated by reference. Examples of cytotoxic moietiesthat can be conjugated to the antibodies include adriamycin,chlorambucil, auristatin E, daunomycin, duocarmycin and analogs thereof,methotrexate, neocarzinostatin, and platinum.

To kill or suppress tumor cells, a first antibody, e.g., an antibody oran antigen binding fragment can be conjugated with a prodrug which isactivated only when in close proximity with a prodrug activator. Theprodrug activator is conjugated with a second antibody, e.g., a secondantibody according to the present invention, preferably one that bindsto a non-competing site on the OV064 molecule. Whether two antibodiesbind to competing or non-competing binding sites can be determined byconventional competitive binding assays. Drug-prodrug pairs suitable foruse in the practice of the present invention are described in Blakely etal., “ZD2767, an Improved System for Antibody-directed Enzyme ProdrugTherapy That Results in Tumor Regressions in Colorectal TumorXenografts,” (1996) Cancer Research, 56:3287-3292, which is herebyincorporated by reference.

Alternatively, the antibody, e.g., the antibody, can be coupled to highenergy radiation emitters, for example, a radioisotope, such as ¹³¹I, aγ-emitter, which, when localized at the tumor site, results in a killingof several cell diameters. See, e.g., S. E. Order, “Analysis, Results,and Future Prospective of the Therapeutic Use of Radiolabeled Antibodyin Cancer Therapy”, Monoclonal Antibodies for Cancer Detection andTherapy, R. W. Baldwin et al. (eds.), pp 303-316 (Academic Press 1985),which is hereby incorporated by reference. Other suitable radioisotopesinclude α-emitters, such as ²¹²Bi, ²¹³Bi, and ²¹¹At, and β-emitters,such as ¹⁸⁶Re and ⁹⁰Y. Moreover, Lu¹¹⁷ may also be used as both animaging and cytotoxic agent.

Radioimmunotherapy (RIT) using antibodies labeled with ¹³¹I, ⁹⁰Y, and¹⁷⁷Lu is under intense clinical investigation. There are significantdifferences in the physical characteristics of these three nuclides andas a result, the choice of radionuclide can be important in order todeliver maximum radiation dose to the tumor. The higher beta energyparticles of ⁹⁰Y may be good for bulky tumors, but it may not benecessary for small tumors and especially bone metastases. Therelatively low energy beta particles of ¹³¹I are ideal, but in vivodehalogenation of radioiodinated molecules is a major disadvantage forinternalizing antibody. In contrast, ¹⁷⁷Lu has low energy beta particlewith only 0.2-0.3 mm range and delivers much lower radiation dose tobone marrow compared to ⁹⁰Y. In addition, due to longer physicalhalf-life (compared to ⁹⁰Y), the tumor residence times are higher. As aresult, higher activities (more mCi amounts) of ¹⁷⁷Lu labeled agents canbe administered with comparatively less radiation dose to marrow. Therehave been several clinical studies investigating the use of ¹⁷⁷Lulabeled antibodies in the treatment of various cancers. (Mulligan T etal. Clin Cancer Res. 1: 1447-1454 (1995); Meredith R F, et al. JNucl Med37:1491-1496 (1996); Alvarez R D, et al. Gynecologic Oncology 65: 94-101(1997)).

The antibodies of the invention can also be conjugated or fused to viralsurface proteins present on viral particles. For example, a single-chainanti-OV064 antibody of the present invention could be fused (e.g., toform a fusion protein) to a viral surface protein. Alternatively, awhole anti-OV064 antibody of the present invention, or a fragmentthereof, could be chemically conjugated (e.g., via a chemical linker) toa viral surface protein. Preferably, the virus is one that fuses withendocytic membranes, e.g., an influenza virus, such that the virus isinternalized along with the anti-OV064 antibody and thereby infectsOV064-expressing cells. The virus can be genetically engineered as acellular toxin. For example, the virus could express or induce theexpression of genes that are toxic to cells, e.g., cell death promotinggenes. Preferably, such viruses would be incapable of viral replication.

The antibodies or antigen binding fragments, of the present inventioncan be used and sold together with equipment, as a kit, to detect theparticular label.

The antibodies, e.g., the antibodies, or antigen-binding portionsthereof, of the present invention bind to extracellular domains of OV064or portions thereof in cells expressing the antigen. As a result, whenpracticing the methods of the present invention to kill, suppress, ordetect cancerous cells, the antibodies or antigen binding fragments,bind to all such cells, not only to cells which are fixed or cells whoseintracellular antigenic domains are otherwise exposed to theextracellular environment. Consequently, binding of the antibodies orantigen binding fragments, is concentrated in areas where there arecells expressing OV064, irrespective of whether these cells are fixed orunfixed, viable or necrotic. Additionally or alternatively, theseantibodies, or antigen binding fragments thereof, bind to and areinternalized with OV064 upon binding cells expressing the antigen.

The anti-OV064 antibodies described herein, e.g., the anti-OV064antibodies, or antigen-binding fragments thereof, may be used incombination with other therapies. For example, the combination therapycan include a composition of the present invention co-formulated with,and/or co-administered with, one or more additional therapeutic agents,e.g., one or more anti-cancer agents, e.g., cytotoxic or cytostaticagents, homone treatment, vaccines, and/or other immunotherapies. Inother embodiments, the anti-OV064 antibodies are administered incombination with other therapeutic treatment modalities, includingsurgery, radiation, cryosurgery, and/or thermotherapy. Such combinationtherapies may advantageously utilize lower dosages of the administeredtherapeutic agents, thus avoiding possible toxicities or complicationsassociated with the various monotherapies.

Administered “in combination”, as used herein, means that two (or more)different treatments are delivered to the subject during the course ofthe subject's affliction with the disorder, e.g., the two or moretreatments are delivered after the subject has been diagnosed with thedisorder and before the disorder has been cured or eliminated. In someembodiments, the delivery of one treatment is still occurring when thedelivery of the second begins, so that there is overlap. This issometimes referred to herein as “simultaneous” or “concurrent delivery.”In other embodiments, the delivery of one treatment ends before thedelivery of the other treatment begins. In some embodiments of eithercase, the treatment is more effective because of combinedadministration. For example, the second treatment is more effective,e.g., an equivalent effect is seen with less of the second treatment, orthe second treatment reduces symptoms to a greater extent, than would beseen if the second treatment were administered in the absence of thefirst treatment, or the analogous situation is seen with the firsttreatment. In some embodiments, delivery is such that the reduction in asymptom, or other parameter related to the disorder is greater than whatwould be observed with one treatment delivered in the absence of theother. The effect of the two treatments can be partially additive,wholly additive, or greater than additive. The delivery can be such thatan effect of the first treatment delivered is still detectable when thesecond is delivered.

When used in combination with one or more additional therapeutic agents,e.g., one or more anti-cancer agents, e.g., cytotoxic or cytostaticagents, homone treatment, vaccines, and/or other immunotherapies, theselected agent or agents depend on the disorder being treated. Theadditional agent(s) may include, for example, a combination withstandard approved therapeutic for the indication being treated (e.g.,platinum (e.g., carboplatin, cisplatin, anthracyclines (e.g.,doxorubicin, epirubicin), taxanes (e.g., paclitaxel, docetaxel),topoisomerase inhibitors (e.g., topotecan, irinotecan, etoposide), vincaalkaloids (e.g., vinorelbine), immunotherapies (e.g., Herceptin),hormone therapy).

Labeled antibodies can be used, for example, diagnostically and/orexperimentally in a number of contexts, including (i) to isolate apredetermined antigen by standard techniques, such as affinitychromatography or immunoprecipitation; (ii) to detect a predeterminedantigen (e.g., in a cellular lysate or cell supernatant) in order toevaluate the abundance and pattern of expression of the protein; (iii)to monitor protein levels in tissue as part of a clinical testingprocedure, e.g., to determine the efficacy of a given treatment regimen

Thus, in another aspect, the present invention provides a diagnosticmethod for detecting the presence of an OV064 protein in vitro (e.g., ina biological sample, such as a tissue biopsy, e.g., from a tumor tissue)or in vivo (e.g., in vivo imaging in a subject). The method includes:(i) contacting the sample with a anti-OV064 antibody or fragmentthereof, or administering to the subject, the anti-OV064 antibody;(optionally (ii) contacting a reference sample, e.g., a control sample(e.g., a control biological sample, such as plasma, tissue, biopsy) or acontrol subject)); and (iii) detecting formation of a complex betweenthe anti-OV064 antibody, and the sample or subject, or the controlsample or subject, wherein a change, e.g., a statistically significantchange, in the formation of the complex in the sample or subjectrelative to the control sample or subject is indicative of the presenceof OV064 in the sample.

Preferably, the anti-OV064 antibody (or fragment thereof) is directly orindirectly labeled with a detectable substance to facilitate detectionof the bound or unbound antibody. Suitable detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials and radioactive materials, as described above and described inmore detail below.

Complex formation between the anti-OV064 antibody and OV064 can bedetected by measuring or visualizing either the antibody (or antibodyfragment) bound to the OV064 antigen or unbound antibody (or antibodyfragment). Conventional detection assays can be used, e.g., anenzyme-linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) ortissue immunohistochemistry. Alternative to labeling the anti-OV064antibody, the presence of OV064 can be assayed in a sample by acompetition immunoassay utilizing standards labeled with a detectablesubstance and an unlabeled anti-OV064 antibody. In this assay, thebiological sample, the labeled standards and the OV064 binding agent arecombined and the amount of labeled standard bound to the unlabeledantibody is determined. The amount of OV064 in the sample is inverselyproportional to the amount of labeled standard bound to the OV064binding agent.

In still another embodiment, the invention provides a method fordetecting the presence of OV064-expressing tumor tissues in vivo. Themethod includes (i) administering to a subject (e.g., a patient having acancer) an anti-OV064 antibody or antigen binding fragment thereof,preferably a antibody or antigen binding fragment thereof conjugated toa detectable label or marker; (ii) exposing the subject to a means fordetecting said detectable label or marker to the OV064-expressingtissues or cells.

Examples of labels useful for diagnostic imaging in accordance with thepresent invention are radiolabels such as ¹³¹I, ¹¹¹In, ¹²³I, ⁹⁹mTc, ³²P,¹²⁵I, ³H, ¹⁴C, and ¹⁸⁸Rh, fluorescent labels such as fluorescein andrhodamine, nuclear magnetic resonance active labels, positron emittingisotopes detectable by a single photon emission computed tomography(“SPECT”) detector or positron emission tomography (“PET”) scanner,chemiluminescers such as luciferin, and enzymatic markers such asperoxidase or phosphatase. Short-range radiation emitters, such asisotopes detectable by short-range detector probes, such as atransrectal probe, can also be employed. The antibody can be labeledwith such reagents using techniques known in the art. For example, seeWensel and Meares (1983) Radioimmunoimaging and Radioimmunotherapy,Elsevier, N.Y., which is hereby incorporated by reference, fortechniques relating to the radiolabeling of antibodies. See also, D.Colcher et al. Meth. Enzymol. 121: 802-816 (1986), which is herebyincorporated by reference.

In the case of a radiolabeled antibody, the antibody is administered tothe patient, is localized to the tumor bearing the antigen with whichthe antibody reacts, and is detected or “imaged” in vivo using knowntechniques such as radionuclear scanning using e.g., a gamma camera oremission tomography or computed tomography. See e.g., A. R. Bradwell etal., “Developments in Antibody Imaging”, Monoclonal Antibodies forCancer Detection and Therapy, R. W. Baldwin et al., (eds.), pp 65-85(Academic Press 1985), which is hereby incorporated by reference.Alternatively, a positron emission transaxial tomography scanner, suchas designated Pet VI located at Brookhaven National Laboratory, can beused where the radiolabel emits positrons (e.g., ¹¹C, ¹⁸F, ¹⁵O, and¹³N).

Fluorophore and chromophore labeled antibodies can be prepared fromstandard moieties known in the art. Since antibodies and other proteinsabsorb light having wavelengths up to about 310 nm, the fluorescentmoieties should be selected to have substantial absorption atwavelengths above 310 nm and preferably above 400 nm. A variety ofsuitable fluorescent compounds and chromophores are described by StryerScience, 162:526 (1968) and Brand, L. et al. Annual Review ofBiochemistry, 41:843-868 (1972), which are hereby incorporated byreference. The antibodies can be labeled with fluorescent chromophoregroups by conventional procedures such as those disclosed in U.S. Pat.Nos. 3,940,475, 4,289,747, and 4,376,110, which are hereby incorporatedby reference.

One group of fluorescers having a number of the desirable propertiesdescribed above is the xanthene dyes, which include the fluoresceinsderived from 3,6-dihydroxy-9-henylxanthhydrol and resamines andrhodamines derived from 3,6-diamino-9-phenylxanthydrol and lissanimerhodamine B. The rhodamine and fluorescein derivatives of9-o-carboxyphenylxanthhydrol have a 9-o-carboxyphenyl group. Fluoresceincompounds having reactive coupling groups such as amino andisothiocyanate groups such as fluorescein isothiocyanate andfluorescamine are readily available. Another group of fluorescentcompounds are the naphthylamines, having an amino group in the α or βposition.

In other embodiments, the invention provide methods for determining thedose, e.g., radiation dose, that different tissues are exposed to when asubject, e.g., a human subject, is administered an anti-OV064 antibodythat is conjugated to a radioactive isotope. The method includes: (i)administering an anti-OV064 antibody as described herein, e.g., aanti-OV064 antibody, that is labeled with a radioactive isotope to asubject; (ii) measuring the amount of radioactive isotope located indifferent tissues, e.g., tumor, or blood, at various time points untilsome or all of the radioactive isotope has been eliminated from the bodyof the subject; and (iii) caluculating the total dose of radiationreceived by each tissue analyzed. The measurements can be taken atscheduled time points, e.g., day 1, 2, 3, 5, 7, and 12, followingadministration (at day 0) of the radioactively labeled anti-OV064antibody to the subject. The concentration of radioisotope present in agiven tissue, integrated over time, and multiplied by the specificactivity of the radioisotope can be used to calculate the dose that agiven tissue receives. Pharmacological information generated usinganti-OV064 antibodies labeled with one radioactive isotope, e.g., agamma-emitter, e.g., ¹¹¹In, can be used to calculate the expected dosethat the same tissue would receive from a different radioactive isotopewhich cannot be easily measured, e.g., a beta-emitter, e.g., ⁹⁰Y.

Deposits

Purified plasmid DNA comprising DNA sequences encoding the heavy andlight chains of each of the human monoclonal antibodies mAb sc77, mAbsc189, and mAb sc209 were deposited on Nov. 10, 2004, on behalf ofMillennium Pharmaceuticals, Inc., 40 Landsdowne St., Cambridge, Mass.02139, U.S.A., at the American Type Culture Collection, P.O. Box 1549,Manassas, Va. 20108, U.S.A., under Accession Nos. PTA-6294 (Ov64sc77),PTA-6295 (Ov64sc189), and PTA-6296 (Ov64sc209). The deposits have beenmade pursuant to, and in satisfaction of, the requirements of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure.

Hybridoma cell lines producing mouse monoclonal antibodies mAb 4G10, mAb8G5, mAb 3A4 and mAb 2F3 were deposited on Dec. 1, 2004, on behalf ofMillennium Pharmaceuticals, Inc., 40 Landsdowne St., Cambridge, Mass.02139, U.S.A., at the American Type Culture Collection, P.O. Box 1549,Manassas, Va. 20108, U.S.A., under Accession Nos. PTA-6402 (4G10),PTA-6403 (8G5), PTA-6401 (3A4), and PTA-6400 (2F3). The deposits havebeen made pursuant to, and in satisfaction of, the requirements of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure.

The present invention is illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference.

EXAMPLES Example 1 Generation of Anti-Ov064 Antibodies andCharacterization

Generation of OV064 Protein for Immunization and Screening:

Ov064 antigen was prepared by subcloning amino acids 32-257(RGSHHHHHHSGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYHTSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQL LNSK (SEQ IDNO:45)) of OV064 (OV064 sequence in bold) into an expression vector suchas pET (Novagen, San Diego, Calif.) and expressed recombinantly in E.coli with a N-terminal 6×-His tag for purification (italic). The proteinwas purified over a Ni-column according to standard protocols asoutlined by the manufacturer (Qiagen, Valencia, Calif.).

For screening of hybridoma supernatants and purified mAbs by ELISA, thenucleic acid encoding the following fusion construct was cloned intopCMV1 expression vector (Sigma):DYKDDDDKLAAANSLIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKL SDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKHHHHHH (SEQ ID NO:46). Purification tags: FLAG-tag (inthe N-terminus) and His-tag (in the C-terminus) were cloned into theconstruct as well and are noted italics. The OV064 sequence amino acids32-257 is depicted in bold. The fusion protein construct was transfectedinto 293 cells, expressed, and recombinant protein was purified over andAnti-FLAG® M2-Agarose Affinity column (Sigma).

Reagents and Cell Lines.

ES-2 ovarian carcinoma cells and SKBR-3 breast carcinoma cells wereobtained from ATCC and maintained according to ATCC protocols. OV064(32-282) was subcloned into pCMV-3 expression vector (Sigma), similar toabove. ES-2_OV064 cells were generated by transfecting OV064 (32-282)into wild type ES-2 cells. Cells were analyzed for OV064 expression byflow cytometry and western blot using the antibodies described below.Goat anti-mouse (GAM)-IgG1-DM1, GAM-IgG2b-DM1, mouse anti-human(MAH)-IgG-DM1, and anti-OV064-DM1 were generated according to a one-stepprocess for the production of cytotoxic conjugates of maytansinoids asdescribed in U.S. Pat. No. 6,441,163.

Briefly, maytansinoids having a disulfide moiety that bears a reactivegroup are linked to cell binding agents, such as antibodies, withoutprior modification of the cell binding agent. Conjugated antibody isprepared by disulfide exchange between the (2-trimethylsilyl)ethyl esterof PPA (5) and N-methyl-N-(3-mercapto-1-oxopropyl)-L-alanine. Preparedantibody is concentrated by tangential flow filtration (10 kD) NMWCOmembranes) and diafiltered against 50 mM potassium phosphate, 2 mM EDTA,pH 6.0. Concentrated antibody is filtered through a filter, ifopalescent, and then modified with N-succinimidyl4-(2-pyridyldithio)propionate (SPP) at a concentration of 20-22 mg/mlantibody and 7 molecules of SPP per molecule of antibody. Modificationis done in 50 mM potassium phosphate, 2 mM EDTA, 5% ethanol, pH 6.0, for2.5+/−0.5 hours. The modification vessel is a 500 ml round bottom flask,modified antibody is separated from the reaction mixture of step 2)using gel filtration chromatography and a Sephadex G25™ column. Thecolumn load represents about 25% of the column volume and thechromatography is done in 50 mM potassium phosphate, 2 mM EDTA, pH 6.0,at a flow rate of 50 cm/hr. The modified antibody elutes between 38-75%column volume. At a concentration of about 10 mg/ml, the modifiedantibody is conjugated with DM1 (using 1.7 molecules of DM1/molecule ofSPP conjugated to the antibodies) for 20+/−4 hours. Typically, thereaction time is between 16.25 and 17.7 hours and is carried out in a 1L round bottom glass flask equipped with a magnetic stirring bar. Theconjugation reaction is done in 3% DMA, 10% sucrose (100 mg sucrose/mlof reaction). At the end of the reaction the conjugated antibody isfiltered and a spectrophotometric reading is taken. Conjugated antibodyis separated from unreacted DM1 by gel filtration chromatography using aSephadex G25™ column.

GAM-IgG1 and GAM-IgG2b were purchased from Southern BiotechnologyCompany. MAH-IgG was purified from clone HP607 (CRL 1753, ATCC).

Generation of Murine mAbs by Immunization:

C57BL6/J mice were immunized with OV064 antigen (see above) and CompleteFreund's Adjuvant (CFA). After two weeks, mice were boosted with OV064antigen and Incomplete Freund's Adjuvant (IFA). Mice continued toreceive booster shots containing antigen every 2 weeks until asignificant antibody titer in the serum of the mice was detected. Uponreaching sufficient titer, mice were sacrificed and spleens wereharvested and homogenized.

Hybridomas that Produce Murine mAb:

Splenocytes from sacrificed C57BL6/J mice were fused to Ag8.35 myelomacells to produce hybridomas and plated in HAT selection medium. Cloneswere screened for mAb production and subcloned by limiting dilution.

Generation of Human mAbs.

XENOMOUSE genetically engineered mice (Abgenix, Fremont, Calif.) (8 to10 weeks old) were immunized for production of human monoclonalantibodies. See, Mendez et al. Nature Genetics 15:146-156 (1997), Greenand Jakobovits J. Exp. Med. 188:483-495 (1998), the contents of whichare incorporated by reference. Briefly, mice were immunizedsubcutaneously at the base of tails with OV064 antigen (see above)emulsified with complete Freund's adjuvant. In each case, the dose wasrepeated three or four times in incomplete Freund's adjuvant. Four daysbefore fusion, the mice received a final injection of OV064 antigen.Spleen and/or lymph node lymphocytes from immunized mice were isolatedfrom the mice and plated in plaque assays as described previously inBabcook et al., Proc Natl Acad Sci USA. 93: 7843-8 (1996), which isincorporated herein by reference. Briefly, cells were plated in agarwith sheep red blood cells, coated with OV064 antigen and cellssecreting mAb against the OV064 antigen would fix complement and lysethe red blood cells immediately surrounding the mAb producing cells.Cells within the cleared plaques were lifted and their immunoglobulinsequences were subcloned into expression vectors. The variable regionsof the heavy and light chains for each of the single cell isolated formAb sc77, mAb sc189 and mAb 209 were cloned as described by the methodsin Babcook et al., Proc Natl Acad Sci USA. 93: 7843-8 (1996). Theselected resulting constructs were cloned into adapted pcDNA3.1(Invitrogen) as described; and constructs encoding these antibodies witha human IgG1 constant region were produced. Supernatants fromtransiently transfected cells containing OV064 specific mAb weresubsequently screened by ELISA and for binding to cells by flowcytometry.

Generation of Human Expression mAb Expression Vectors.

Each of the heavy and light chain variable regions for mAb sc77, mAb sc189 and mAb 209 was then cloned into an expression vector comprisingboth the heavy and light chain of each mAb. Briefly, primers weredesigned with specified cloning sites for PCR amplification of heavy andlight chain sequences from the original vectors: 3′ VH primers have Blp1cloning site (underlined); 5′ VH primers have EcoR1 cloning site(underlined); 3′ VL primers have BsiW1 cloning site (underlined); and 5′VL primers have NotI cloning site (underlined):

sc077 3′ VH primer: (SEQ ID NO: 31)AAAAAGAGAGCTGAGCTGACGGTGACCAGGGTTCCCTGG sc077 5′ VH primer:(SEQ ID NO: 32) AAAAACTCTGAATTCCTCACCATGGAGTTGGGACTGTGT sc077 3′VL primer: (SEQ ID NO: 33) AAAAAGAGACGTACGTTTGATCTCCACTTTGGTCCCTCCsc077 5′ VL primer: (SEQ ID NO: 34)AAAAACTCTGCGGCCGCCTCACCATGGTGTTGCAGACCCAGGTC sc189 3′ VH primer:(SEQ ID NO: 35) AAAAAGAGAGCTGAGCTGACGGTGACCAGGGTTCCCTGG sc189 5′VH primer: (SEQ ID NO: 36) AAAAACTCTGAATTCCTCACCATGGAATTTGGACTTCGCsc189 3′ VL primer: (SEQ ID NO: 37)AAAAAGAGACGTACGTTTAATCTCCAGTCGTGTCCCTTG sc189 5′ VL primer:(SEQ ID NO: 38) AAAAACTCTGCGGCCGCCTCACCATGAGGGTCCCTGCTCAG sc209 3′VH primer: (SEQ ID NO: 39) AAAAAGAGAGCTGAGCTGACGGTGACCGTGGTCCCTTGGsc209 5′ VH primer: (SEQ ID NO: 40)AAAAACTCTGAATTCCTCACCATGGAGTTGGGGCTGTGC sc209 3′ VL primer:(SEQ ID NO: 41) AAAAAGAGACGTACGTTTGATTTCCACCTTGGTCCCTTG sc209 5′VL primer: (SEQ ID NO: 42) AAAAACTCTGCGGCCGCCTCACCATGGAAACCCCAGCGCAG.5′ primers also contain a Kozak sequence in front of the ATG of thenative human signal sequence of the antibodies. PCR was done withInvitrogen's Platinum Pfx DNA Polymerase (highest fidelity) cat. no.11708-021 to amplify each of the human sequences. The resulting PCRproducts were digested and light chain variable region genes were clonedinto the expression vector, pLKTOK58D, followed by insertion of theheavy chain variable region genes.

Expression vector pLKTOK58D contains an expression cassette, the designand generation of which have been described previously. See O'Keefe etal., U.S. Patent Application Publication No. 20040033561, published Feb.19, 2004, which is incorporated herein by reference. Briefly, pLKTOK58Dis a pcDNA3 based expression vector, which was engineered to replace theCMV promoter with an EF-1a promoter. The MfeI restriction site in theEF1a promoter was mutated by site directed mutagenesis. Thisintermediate expression vector also contains the BGH polyadenylationsite following a multiple cloning site downstream of the promoter.Additionally, the DHFR resistance cassette with its own SV40 promoterand polyadenylation site was inserted into the expression vector tocreate pTOK10. The human heavy chain IgG1 constant region gene wasinserted into the multiple cloning site between the EF1a promoter andthe BGH polyadenylation site, resulting in a promoter, heavy chain,polyA cassette, with a cloning site allowing addition of variablesequence, creating pLKTOK55. Similarly, human light chain kappa constantregion gene was engineered to introduce a silent BlpI mutation, and alsoinserted into the intermediate expression vector pLKTOK10. The entireregion of promoter through polyA site of each of the heavy chain andlight chain were combined in one vector, resulting in pLKTOK58D (seeFIG. 1). The light chain cassette has cloning sites allowing forinsertion of variable sequences in frame into NotI/BsiWI restrictionsites, and the heavy chain cassette has cloning sites allowing forinsertion of variable sequences in frame into EcoRI/BlpI restrictionsites.

The resulting expression vectors contain each of the full length heavyand light chain genes under the control of the EF-1 alpha promoter. Bothheavy and light chains also have BGH polyadenylation sites at their 3′ends. The vector also has two selection genes, neomycin anddihydrofolate reductase, for selection in transfected cell line ofchoice, such as, for example, CHO cells. The ampicillin gene is also inthe expression vector for bacterial selection and growth, preferably,for example, an endonuclease and recombination negative strain (endA,recA) such as DH5alpha. Resulting expression vectors for each of thehuman antibodies mAb sc77 (Ov64sc77), mAb sc189 (Ov64sc189), and mAbsc209 (Ov64sc209) were deposited with the American Type CultureCollection as described above.

Analysis of mAb by ELISA.

Microtiter plates were coated with 50 ul of recombinant OV064 in PBS atconcentration of 1 μg/ml. Plates were covered with film and incubatedovernight at room temperature, antigen was decanted from plates, andplates washed with phosphate buffered saline+1% Tween20. 200 ul per wellof blocking solution (10% Nonfat dry milk in phosphate buffered saline)was added and incubated 1 hr at room temperature. 50 ul test reagent(either hybridoma supernatants, mouse serum or purified mAb) was addedto the plate and incubated at room temperature. After 1 hr, plates werewashed 3 times with phosphate buffered saline+1% Tween20, followed byaddition of a working concentration of detection Ab (Anti-mouse orAnti-human horseradish peroxidase conjugate), usually diluted 1:5000 to1:10000 in blocking buffer and incubated at room temperature. After 1hr, plates were washed 3 times with phosphate buffered saline+1%Tween20. Bound anti-OV064 mAb was visualized with3,3,5,5′-tetramethylbenzidine (TMB) Substrate, and the reaction stoppedwith 2M H₂SO₄. Plates were read at 450 nm.

Analysis of mAb by Flow Cytometry.

50,000-100,000 test cells were prepared in 100 ul phosphate bufferedsaline+1% fetal bovine serum (PBSS). 50 ul of test reagent (eitherhybridoma supernatants, mouse serum or purified mAb) was added, andincubated 30-45 min on ice, then washed 2-3 times in PBSS. 50 ul offluorescently labeled secondary antibody (Anti-Mouse IgG or Anti-HumanIgG) was then added, and incubated 30-45 minutes on ice and washed. Thecells were then resuspended in 300 ul of PBSS and analyzed by flowcytometer (FACSCalibur, Becton Dickinson) to assess amount of cellsurface bound antibody.

PI-PLC Treatment of GPI Linked Cell Surface Proteins.

Determination of release of GPI-linked proteins from cultured cells, wasassessed using a 60 mm diameter culture dish, containing approximately0.5.1×10⁶ cells. The cell culture was rinsed twice with coldphosphate-buffered saline (PBS), then 0.5 mL of the same buffercontaining 0.1.1.0 units of B. cereus Phosphatidylinositol-specificphospholipase C(PI-PLC) was added, and rocked at 4° for 20 minutes.Buffer was recovered for analysis of released proteins by western blot.

Internalization Assay.

Cells were plated in duplicate microtiter plates in complete medium andallowed to grow to about 40-50% confluency. Growth medium was removedand cells were placed on ice in PBSS. mAb was added to each well andincubated for 30 minutes on ice. Cells were washed 3 times in PBSS andafter final wash 100 ul of PBSS were added. One of the plates was placedat 37 C and the other plate was left on ice and incubated for 1 hour.Cells were fixed in 4% formaldehyde and permeabilized in 1% TritonX-100, both in PBS. Working solutions of Fluorescently labeled secondaryantibody (Anti-mouse IgG or Anti-Human IgG) was added to each well andincubated for 30 in on ice. Fluorescence was detected by microscopy.

Cytotoxicity Assays.

The ability of toxin conjugated anti-OV064 mAbs to kill OV064 expressingcells was tested. 1000-1500 cells were plated in complete medium(DMEM+10% FBS) and various concentrations of unconjugated, or DM1 or DM4conjugated primary OV064 specific mAb. The ability of unconjugatedprimary OV064 specific mAb to kill OV064 expressing cells when bound toa DM1 or DM4 conjugated secondary antibody was also tested. Variousconcentrations of primary unconjugated OV064 specific mAbs were added tocells together with 5-10 nM of a relevant secondary DM1 conjugatedantibody (Goat anti-mouse IgG or Mouse anti-human IgG). Cells wereincubated for 96 hours. Cell death was measured by adding WST cellviability reagent. After 1-2 hour development the optical density in thewells was read at 650 nM.

Epitope Mapping.

A variety of techniques are available to determine epitopes specific forany particular antibody. For example, constructs of OV064 truncationswere generated in a pCMV3 vector. Constructs representing amino acids32-282, 133-282, 167-282 and 183-282 of the OV064 protein sequence weregenerated as N-terminally FLAG tagged proteins that when transfectedinto 293 cells, will be expressed on the cell surface. OV064 specificantibodies sc077, sc189, sc209 an 8G5 were tested for binding to theseexpressed constructs by flow cytometry, as described above. Furthermore,epitopes were analyzed using the Novatope system (Novagen Inc). Briefly,OV064 cDNA was treated with DNAse and fragments representing 15-600 bpwere cloned into a bacterial expression vector. Each mAb was testedagainst 2000 clones representing OV064 fragments and each positive clonewas picked and sequenced. The minimal common sequence between each ofthese clones was designated as the epitope to which each antibody bound.Additionally, a synthetic peptide map was utilized for identifyinganti-OV064 epitopes. Briefly, 20 amino acid peptides each overlappingwith 15 amino acids were printed on a nitrocellulose filter andincubated with anti-OV064 specific antibodies. Peptide spots thatreacted strongly with the OV064 specific antibodies indicated thepresence of an OV064 epitope.

Results:

Mouse monoclonal antibodies mAb 4G10, mAb 8G5, mAb 3A4 and mAb 2F3, weregenerated by traditional hybridoma technology. Human monoclonalantibodies sc77, sc189 and sc209 were generated using transgenic micethat generate fully human IgG1 antibodies, utilizing Abgenix XENOMAXtransgenic technology. Specificity of the antibodies against OV064 wastested by ELISA and flow cytometry (FCM). A subset of the generatedantibodies was selected for further characterization.

Hybridoma cell lines producing mouse monoclonal antibodies mAb 4G10, mAb8G5, mAb 3A4 and mAb 2F3 were deposited on Dec. 1, 2004, on behalf ofMillennium Pharmaceuticals, Inc., 40 Landsdowne St., Cambridge, Mass.02139, U.S.A., at the American Type Culture Collection, P.O. Box 1549,Manassas, Va. 20108, U.S.A., under Accession Nos. PTA-6402 (4G10),PTA-6403 (8G5), PTA-6401 (3A4), and PTA-6400 (2F3).

The results of sequence analysis for each of the human monoclonalantibodies mAb sc77, mAb sc189, and mAb sc209 are listed in the sequencelisting, and summarized in Table 1. Each of the sequences for nucleicacid (NA) and amino acid (AA) are described. The amino acid sequencesfor each of the heavy and light chains for the human monoclonalantibodies mAb sc77, mAb sc189 and mAb 209 is shown in Table 2.

TABLE 1 Summary of sequences for human monoclonal antibodies. mAb IgGchain NA SEQ ID AA SEQ ID Sc77 Heavy chain  1  2 Light chain  3  4 Sc189Heavy chain  5  6 Light chain  7  8 Sc209 Heavy chain  9 10 Light chain11 12

TABLE 2 Amino acid sequence of human mAb SEQ IgG ID mAb chain NO:Amino Acid Sequence 1 Sc77 Heavy  2MELGLCWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSC chainAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTSGIAAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 2 Sc77 Light  4MVLQTQVFISLLLWISGAYGDNVMTQSPDSLAVFLGERATINC chainKSSQSVLYNSNYKNYLAWYQQKPGQPPKLLFYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKSFNRGEC 3Sc189 Heavy  6 MEFGLRWVFLVAILEGVQCEVQLVESGGGLVQPGGSLRLSCA chainASGFSFSSYDMHWVRQATGKGLEWVSGIDIAGDTYYPGSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCARGDYDGTFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSREDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4 Sc189 Light  8MRVPAQLLGLLMLWVPGSSGDIVMTQTPLSSPVTLGQPASISC chainRSSQSLVHSDGNTYLSWLQQRPGQPLRLLFYKISNRFSGVPDRFSGSGAGTDFTLIINRVEAEDVGVYYCMHATQFPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 5Sc209 Heavy 10 MELGLCWLFLVAILEGVQCGVQLVESGGGLVQPGGSLRLSCA chainASGFTISRNDMHWVRQATGKGLEWVSAIGTGGDTYYPGSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCARGHMTTFGGFIVIGNGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 6 Sc209 Light  12METPAQLLFLLLLWLPDTTGEIVMTQSPATLSVSPGERATLSCR chainASQSVRSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYSCQQYNNWPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

Table 2, row 1 is an illustration of the encoded amino acid sequence ofthe mature heavy chain variable region of mAb sc77 (SEQ ID NO: 2); andthe nucleic acid sequence encoding the mature heavy chain variableregion of mAb sc77 is depicted in SEQ ID NO:1. Complementaritydetermining region (CDR) 1 consists of amino acid residues 50-54 of SEQID NO: 2 (SEQ ID NO: 13), CDR 2 consists of amino acid residues 69-85 ofSEQ ID NO: 2 (SEQ ID NO: 14), CDR 3 consists of amino acid residues118-126 of SEQ ID NO: 2 (SEQ ID NO: 15).

Table 2, row 2 is an illustration of the encoded amino acid sequence ofthe mature light chain variable region of mAb sc77 (SEQ ID NO: 4); andthe nucleic acid sequence encoding the mature kappa light chain variableregion of mAb sc77 is depicted in SEQ ID NO: 3. Complementaritydetermining region (CDR) 1 consists of amino acid residues 44-59 of SEQID NO: 4 (SEQ ID NO: 22), CDR 2 consists of amino acid residues 76-82 ofSEQ ID NO: 4 (SEQ ID NO: 23), CDR 3 consists of amino acid residues115-123 of SEQ ID NO: 4 (SEQ ID NO: 24).

Table 2, row 3 is an illustration of the encoded amino acid sequence ofthe mature heavy chain variable region of mAb sc 189 (SEQ ID NO: 6); andthe nucleic acid sequence encoding the mature heavy chain variableregion of mAb sc77 is depicted in SEQ ID NO:5. Complementaritydetermining region (CDR) 1 consists of amino acid residues 50-54 of SEQID NO: 6 (SEQ ID NO: 16), CDR 2 consists of amino acid residues 69-84 ofSEQ ID NO: 6 (SEQ ID NO: 17), CDR 3 consists of amino acid residues117-125 of SEQ ID NO: 6 (SEQ ID NO: 18).

Table 2, row 4 is an illustration of the encoded amino acid sequence ofthe mature light chain variable region of mAb sc189 (SEQ ID NO: 8); andthe nucleic acid sequence encoding the mature kappa light chain variableregion of mAb sc77 is depicted in SEQ ID NO: 7. Complementaritydetermining region (CDR) 1 consists of amino acid residues 44-59 of SEQID NO: 8 (SEQ ID NO: 25), CDR 2 consists of amino acid residues 75-82 ofSEQ ID NO: 8 (SEQ ID NO: 26), CDR 3 consists of amino acid residues114-122 of SEQ ID NO: 8 (SEQ ID NO: 27).

Table 2, row 5 is an illustration of the encoded amino acid sequence ofthe mature heavy chain variable region of mAb sc209 (SEQ ID NO: 10); andthe nucleic acid sequence encoding the mature heavy chain variableregion of mAb sc77 is depicted in SEQ ID NO:9. Complementaritydetermining region (CDR) 1 consists of amino acid residues 50-54 of SEQID NO: 10 (SEQ ID NO: 19), CDR 2 consists of amino acid residues 69-84of SEQ ID NO: 10 (SEQ ID NO: 20), CDR 3 consists of amino acid residues117-134 of SEQ ID NO: 10 (SEQ ID NO: 21).

Table 2, row 6 is an illustration of the encoded amino acid sequence ofthe mature light chain variable region of mAb sc209 (SEQ ID NO: 12); andthe nucleic acid sequence encoding the mature kappa light chain variableregion of mAb sc77 is depicted in SEQ ID NO: 11. Complementaritydetermining region (CDR) 1 consists of amino acid residues 44-54 of SEQID NO: 12 (SEQ ID NO: 28), CDR 2 consists of amino acid residues 70-76of SEQ ID NO: 12 (SEQ ID NO: 29), CDR 3 consists of amino acid residues109-117 of SEQ ID NO: 12 (SEQ ID NO: 30).

Expression vectors were created as described above which contain codingsequence for both the heavy and light chain of each of mAb sc77, mAbsc189, and mAb sc209. Purified DNA encoding the heavy and light chainsof each of the human monoclonal antibodies the lumen of the ER. Toinvestigate whether Ov064 is a GPI-linked protein, cells expressing(ES-2_O V064) and cells not expressing (ES-2 wt) were treated withphosphatidylinositol-specific phospholipase (PI-PLC), a commonly usedGPI anchor—cleaving enzyme, which releases GPI linked proteins into thesupernatant. The presence of Ov064 was then analyzed by westernblotting. OV064 was not detected in the supernatant of control treatedES-2_OV064 cells. In contrast, upon PI-PLC treatment Ov064 was readilydetected in the supernatant, thus providing evidence that Ov064 is aGPI-linked cell surface protein.

To investigate whether mAbs to Ov064 would internalize in an antigenspecific manner, cells expressing Ov064 (ES-2_OV064) were preincubatedon ice with saturating concentrations of mAb. Excess mAb was washed awaybefore cells were incubated for 1 hour at 37° C. Intracellular IgG wasdetected by immunofluorescence to visualize internalization. For each ofmAb 4G10, mAb 8G5, mAb 3A4 and mAb 2F3, cells which were kept on ice,all of the Ov064 mAb was found on the cell surface of the cells and nointracellular mAb was detected. However when cells were incubated at 37°C., cytoplasmic IgG was readily detected internally for each of mAb4G10, mAb 3A4, mAb 2F3 and mAb 8G5. This established that the antibodyhad been transported across the membrane most likely in complex withOv064. No cell surface binding or internalization was seen in ES-2_Neoor ES-2 wt cells.

The ability of mAbs to Ov064 to deliver cytotoxic agents was assessed.Various concentrations of OV064 mAb were added to cells in microtiterplates, either alone as unconjugated mAb, together with DM1 conjugatedanti-mouse IgG or anti-human IgG; or as directly DM1 conjugated mAb andincubated for 4 days as described in the Methods. Cell viability ofOV064 expressing cells was determined and plotted against control cellsthat were not recognized by antibody. Anti-OV064 antibody alone does nothave any effect on cell viability and neither does similar concentrationof control antibody MAH-IgG-DM1.

TABLE 4 Cytotoxic effects of human IgG and mouse IgG mAbs specific forOV064. ICA FCM ELISA IgG (EC50) (MFI max) (EC50) Isotype sc77   0.123 nM780 ND Human IgG1 sc189    0.06 nM 820 ND Human IgG1 sc209   0.053 nM700 ND Human IgG1 4G10      3 nM 350  0.1 μg/ml Mouse IgG1 3A4     12 nM374 0.02 μg/ml Mouse IgG1 2F3    0.66 nM 138 0.03 μg/ml Mouse IgG2b 8G5  >100 nM 380 0.05 μg/ml Mouse IgG1

However, when anti-Ov064 antibody was used in conjunction with the DM-1conjugated anti-mouse IgG, cell killing was observed, resulting in EC50of approximately 0.6-12 nM of the OV064 mAb. See Table 4. No cellkilling was observed on cells that do not express OV064 that weretreated with the same combination of mAb and toxin conjugated secondaryantibody. Each of the murine and human mAbs were shown to bind to OV064by ELISA and flow cytometry (FCM). See Table 3 and Table 4. Whencompared in cytotoxicity assays, however, there were some unexpecteddifferences observed between the different mAb. Each of the mouse IgG4G10, 3A4 and 2F3 and human IgG sc77, sc189, and sc209 could efficientlyinternalize and deliver toxic payloads into OV064 expressing cells,resulting in cell killing. mAb 3A4 was shown to internalize in cells butrequired higher concentrations of the mAb to demonstrate identicallevels of cytoxic effects. In contrast, while mouse IgG 8G5 was able tobind with high affinity to recombinant protein and to cells expressingOV064, the mAb was completely unable to bring toxic payload into OV064expressing cells. Thus, while each of the described mouse and human IgGmolecules binds to OV064, and may be useful for detection of OV064expression, only a subset of mAb specific for Ov064 are effective indelivery of toxic payloads into OV064 expressing cells. Similar resultswere found using primary OV064 mAbs that were directly conjugated withDM1. This effect does not appear to relate to affinity, as not all mAbsthat bind with high affinity to OV064 could function as an effectiveimmunoconjugate for killing tumor cells.

As described above in the methods, various approaches to determineepitope mapping of OV064 specific mAbs were used. Table 5 demonstratesresults of the peptide binding experiments, which revealed sc77 binds anepitope comprising amino acids in the region of amino acids 167-176 ofOV064; sc189 binds an epitope comprising amino acids in the region ofamino acids 238-257 of OV064; sc209 binds an epitope comprising aminoacids in the region of amino acids 177-181 of OV064, and 8G5 binds anepitope comprising amino acids in the region of amino acids 67-76 ofOV064. The other methods confirmed the results depicted in Table 5.

TABLE 5 OV064 Epitope Mapping of IgG mAbs OV064 AA sc77 sc189 sc209 8G532-51 − − − − 37-56 − − − − 42-61 − − − − 47-66 − − − − 52-71 − − − −57-76 − − − +++ 62-81 − − − +++ 67-86 − − − +++ 72-91 − − − − 77-96 − −− −  82-101 − − − −  87-106 − − − −  92-111 − − − −  97-116 − − − −102-121 − − − − 107-126 − − − − 112-131 − − − − 117-136 − − − − 122-141− − − − 127-146 − − − − 132-151 − − − − 137-156 − − − − 142-161 − − − −147-166 − − − − 152-171 − − − − 157-176 +++ − + − 162-181 +++ − + −167-186 +++ − +++ − 172-191 + − +++ − 177-196 − − +++ − 182-201 − − − −187-206 − − − − 192-211 − − − − 197-216 − − − − 202-221 − − − − 207-226− − − − 212-231 − − − − 217-236 − − − − 222-241 − − − − 227-246 − − − −232-251 − − − − 237-256 − +++ − − 238-257 − +++ − −

Example 2 Anti-OV64 ADC Selectively Blocks Antigen Expressing Cells inG2/M

Materials and Methods.

ES-2, ES-2_OV64 and SKBR3 human tumor cells were plated on 6 well dishes(2×10⁵/well; Falcon). Cells were cultured in McCoy's 5A medium (Gibco)supplemented with 10% bovine calf serum and 2 mM L-glutamine. After 18hours, 209-DM4 or 209 was added directly to the cells at variousconcentrations (1.15, 2.5 and 6.5 nM) and the cells were grown anadditional 6, 24 or 48 hrs. Cells treated with DMSO (0.2%) served as theuntreated vehicle control and those treated with taxol or vincristine(100 nM) as the control for mitosis. The cells were harvested withTrypsin EDTA 1× (Gibco), washed 1× with phosphate buffered saline (PBS),fixed in 70% ethanol and stored at 4° for 1 hr. The cells were thenresuspended in propidium iodide (1:40, Molecular Probes) and RNAse A(1:5000, Sigma) in PBS for 30 min at 4° C. Cell cycle distributions weredetermined by measuring DNA content using flow cytometry (FACSCalibur;Becton Dickenson) and samples were analyzed using Winlist software(Verity).

Results.

To examine the affect of 209 and 209-DM4 on the cell cycle, DNA profileswere evaluated by flow cytometry. In the ES-2 cells overexpressing Ov64,treatment with 6.5 nM 209-DM4 for 24 hours induced an increase in 4Ncells relative to control cells. At 48 hours a subG2 and subG1 scatterof cells was observed indicating apoptosis. In contrast ES-2 wt treatedwith 6.5 nM 209-DM-4 were similar to control at both 24 and 48 hoursindicating that overexpression of Ov64 is necessary and responsible forsensitivity to 209-DM4. An increase in 4N cells upon treatment with209-DM4 is consistent with inhibition mediated by DM4, a microtubulebinder which is known to abrogate cell cycle progression at mitosis (4NDNA content) Both cell lines responded to treatment of 100 nM taxol withan equivalent increase in 4N cells indicating that the ES-2 cells areequally responsive to mitotic inhibitors. At 48 hrs the SKBR3 cells thatexpress Ov64 endogenously demonstrated an increase in 4N cells.

Example 3 In Vitro Cytotoxicity of Anti-OV64 ADC

Materials and Methods.

ES-2 wt and ES-2_OV64 cells were cultured to about 70-80% confluency andlifted with Versene and live cells were counted by Trypan Blueexclusion. Cells were collected in cold D-PBS or D-PBS/3% FBS/0.1%sodium azide buffer and resuspended in McCoy's/10 FBS (complete medium)to 2×10⁵ cells/mL. Add 100 μl, of cells suspension was added to eachwell of a 96-well falt bottomed plate (2000 cells per well). SKBR-3cells were treated the same way except that 7500 cells were plated perwell. The anti-OV64 ADC sc209-DM4 was diluted in complete medium in a2-fold serial dilution. As controls, no ADC in complete medium was used.100 μL of ADC dilutions was added to the cells in triplicate. The cellswere incubated at 37° C., 5% CO2 for 96 hours. At the end of theincubation time the media was aspirated and a 10% WST in complete mediawas added. Plates were incubated at 37° C. for 1.5-2 hours and read onplate reader at OD 440/650.

Results.

The cytotoxic effect of sc209-DM4 on ES-2WT and ES-2_Ov64 cells wascalculated as percent of control (no ADC added) cells. The LD50 forsc209-DM4 on ES-2_Ov64 cells is 2.5 nM whereas the LD50 for the ES-2WTcells was calculated to be >25 nM as calculated in ExcelFit.

Both the ES-2WT and ES-2_OV64 cells have similar sensitivity to freemaytanisinoids (data not shown). However when DM4 is conjugated tosc209, the ES-2_OV64 cells are >10 fold more sensitive than the ES-2WT.Thus DM4 has been rendered target selective by conjugation to sc209 andselectively kill ATGOv064 expressing cells.

Although the level of ATGOv064 expression is significantly lower on theSKBR3 cells, the sensitivity to sc209-DM4 is not significantlydifferent. The LD50 for SKBR3 was 4.4 nM (n=3) compared to 2.5 nM onES-2_OV64. As a control for a non-binding DM4 conjugated antibody,SKBR-3 cells were incubated with 5H9-DM4, which does not bind to theSKBR3 cells.

Example 4 Radiolabeled Anti-Ov064 Antibody Selectively Localizes toAntigen Expressing Tumors In Vivo

Materials and Methods

DTPA Conjugation.

Antibodies were reconstituted in conjugation buffer (0.1 M sodiumbicarbonate, pH 8.2) by Contricon (YM-30, Milipore) to obtain a 5 mg/mlsolution of antibodies by ultrafiltration (Centricon® filter 30 kDa,Millipore at 5000 g). The chelator diethylenetriamine pentaacetic aciddianhydride (DTPA dianhydride, Sigma Chemical Co., St Louis, Mo.,U.S.A.) was then conjugated to the antibody using a small modificationof the well-known cyclic anhydride method. The conjugation condition wasperformed and optimized with human polyclonal immunoglobulin G (HIG,Sigma Chemical Co., St Louis, Mo., U.S.A.) at antibody: DTPA molarratios of 1:1 to 1:50. The ratio of 1:10 was used for the laterexperiments. In brief, 5 μl of a 2.3 mg/ml suspension of DTPA anhydridein dry DMSO (Sigma-Aldrich) was added dropwise to test tubes containing2 mg/0.4 ml antibody. The reaction was gently mixed and incubated atroom temperature for 50 min. Unbound DTPA was then removed byultrafiltration (10 times 50 min at 4500×g). At end of the purification,the Ab solution was changed into labeling buffer (0.1 M sodium acetatebuffer, NaOAc, pH 7.2). The purified immunoconjugate was measured forthe protein concentration and dispensed into 160 ug/100 ul, eitherstored at −80° C. or immediately used for radiolabelling.

Radiolabelling.

¹¹¹I, as ¹¹¹InCl₃, was purchased from PerkinElmer, Boston in 370 MBq/mlin 0.05 M HCl, pH 1.5-1.9. The ¹¹¹InCl₃ was added to the conjugatedDTPA-Ab and the reaction mixture was incubated for 5 min. The productwas diluted in normal saline to the desired specific activity. After theLE and RCP were measured, the resulting ¹¹¹In-DTPA was sterilised byfiltration through a 0.2 μm syringe filter (Millipore).

The complete labelling procedure was optimised by subsequently varyingreaction pH, incubation times, molar DTPA to antibody ratios, amounts of¹¹¹InCl₃ added per mg conjugate and QC methods with a Sephadex G25column (PD-10 column, Amersham Biosciences AB, Uppsala, Sweden) versusITLC. Initially, purification methods with a Sephadex G25 column (PD-10column, Amersham Biosciences AB, Uppsala, Sweden) versus ultrafiltrationwas planned to removed the unlabeled ¹¹¹In. Then, the one-step instantlabeling method was developed, which can achieve the LE up to 97%, andthe RCP remains that level 5 days after the labeling. The labeling canbe achieved by a simple addition of required amount of ¹¹¹InCl₃ to thevials containing known amount of immunoconjugates.

Dual-Tumor Animal Model.

The parental ES-2 human ovarian cell line and the Ov64 transfected ES-2line (ES-2Ov64) were grown in culture at 37° C. in a humidified 5% CO₂incubator in McCoy 5a medium with 10% FBS. Xenografts were establishedin 25 gram (g) female BALB/c nu/nu mice, 8-10 weeks of age with theimplantation of 2×10⁶ ES-2 or ES-2Ov64 cells into the left and rightrear dorsum subcutis, respectively. In a pilot study, tumor growthkinetics of the two tumors grown in the same animal were evaluated bymeasuring the tumor sizes over time and the two tumors shared similargrowth kinetics (data not shown).

Tumor growth was monitored with caliper measurements; tumor volume wascalculated using the formula V=W²×L/2. Animals were randomized into 3groups of 30 animals (5 animals per timepoint) with tumor sizes of100-300 mg (5-8 mm diameter) on both tumors.

Biodistribution and Tumor Uptake Studies.

Tumor-bearing mice were injected i.v. via a lateral vein with 0.1 ml of¹¹¹In-Abs, specifically, SC189, SC209 and the control HIG at 300 μCi/15μg/100 μl. Ninety animals were injected as: 5 (animals per group)×3antibodies×6 timepoints (5 min, 4, 24, 48, 120 and 168 h). At the presettime points after injection, mice were sacrificed by CO₂. Blood wasdrawn by cardiac puncture immediately after euthanasia, and thefollowing tissues were obtained by dissection: tumors, liver, kidneys,spleen, lungs, heart, stomach, small intestines, large intestines,muscle, pancreas, and ovaries. Tissues were weighed, and theradioactivity was measured using an automatic gamma counter with decaycorrection (PerkinElmer). Aliquots of the dose solution were alsocounted to determine the total radioactivity administered. The countswere then standardized for tissue mass and expressed as a percentage ofthe total activity injected into each mouse per gram tissue (% ID/g).

Whole Body Clearance.

In the first batch of pilot study, the whole-body clearance wasmeasured. After injection of control HIG or Sc209, the whole bodyradioactivity was measured by placing the animal into a dose calibrator.While clinical PK is measured by the elimination, we measure theretention of radioactivity. With periodic measurements, we can calculatethe effective half-life of the radiolabeled antibodies (T_(1/2 eff)).The biological halflife (T_(1/2 bio)) can be computed by the followingequation: 1/T_(1/2 eff)=1/T_(1/2 bio)+1/T_(1/2 phy), where T_(1/2 phy)is the physical half-life of the radiolabel (67 hrs for 111In). Animalswere evaluated for whole body radioactivity at the following timepoints:0, 4, 24, 48, and 120 hrs.

Imaging.

One of 5 euthanized animals from each group at each time point wasimaged anteriorly with a bench-top scintillation camera (Gamma Imager,Biospace, France) with a medium energy collimator.

Autoradiography.

After imaging, one euthanized mouse from each group was subjected towhole-body autoradiography to reveal the interior distribution oflabeled Ab and to compensate for the insufficient spatial resolution ofthe planar scintigraphic imaging. Briefly, immediately after imaging,animals were snap frozen by dry-ice-cooled ethanol, then embedded in aframe containing 4% Carboxymethyl cellulose in water (Sigma-Aldrich).The embedded animals were frozen in −80° C. overnight shielded withlead. Then the sample (whole body) was mounted on an existingcryomicrotome and brought to thermal equilibrium (−20° C.) for at least2 hrs. The block was then sliced into coronal sections 40 μm thick.Sections were immobilized on tape (Type 810, Leica Inc., Deerfield, Ill.K) and left in the instrument to freeze-dry. After 8 hrs, the tapes withtissues were set in contact with a Phosphor screen (Fuji imaging) andexposed for 72 hrs, then scanned in a PhosphorImager Fuji scanner togenerate a digitized image.

Results

This study was performed to determine the in vivo biodistribution of theOv64 Sc209 and Sc 189 human monoclonal antibodies by imaging tumorbearing mice. The Ov64 antibodies and a control polyclonal humanimmunoglobulin G (HIG) were radiolabeled with 111In using DTPA as abifunctional chelator. The in vivo behavior, including tumor targetingand biodistribution in normal tissues over time, was investigated with amurine dual-tumor model with both Ov64(−) and Ov64(+) tumors. In vivogamma imaging (scintigraphy) and whole-body autoradiography (WBA) wereacquired, and tissue radioactivity counting was used to supplement thespatial resolution. Both anti-Ov64 antibodies were with 111In with aone-step procedure. The labeling efficiency is higher than 95% whichmakes post-labeling purification unnecessary. The DTPA-conjugatedantibodies are stable at least 10 weeks at −80° C. and are stable atleast 120 hrs after labeling. Both sc189 and sc209 demonstratedselective targeting to antigen expressing tumors and only backgroundlevels of antibody were found in the corresponding antigen negativetumor in the same animal. This differential tumor targeting wasvisualized by noninvasive scintigraphy, and validated by ex vivo WBA andtissue counting biodistribution. The tumor exposure for SC 189 was 991nM·h and that number for SC209 was 709 nM·h when 15 μg labeled antibodywas injected into a 25 g mouse (0.6 mg/kg).

Conclusions:

The Sc189 and Sc209 antibodies have high specificity for the Ov064expressing ovarian cancers. Imaging technology using labeled anti-Ov064antibodies is a powerful tool for in vivo tumor detection(Radioimmunoimaging) and staging.

Example 5 Anti-OV64 ADC Shrinks Antigen Expression Tumors In Vivo

Materials and Methods.

Eleven week old female nude mice (Taconic Farms, Inc) were inoculatedwith 2×10⁶ ES2/OV64 cells in the sub cutis and tumor growth wasmonitored twice weekly with caliper measurements. The mean tumor volumewas calculated using the formula V=W²×L/2. When the mean tumor volumereached approximately 200 mm³, mice were then randomized into treatmentgroups containing 10 mice per group. Mice were dosed I.V. (200 μL dosingvolume) twice weekly for a total of 5 doses. The dosing solutions wereprepared immediately prior to dosing. The dosing solutions for209-SPDB-DM4 (Lot #2124-109), L-DM4 (Lot#02-058-36-16) and Sc209antibody were prepared in phosphate buffered saline as the dosingvehicle.

The treatment groups included: vehicle, 300 μg/kg DM4 toxin, 12.4 mg/kgSc209 naked antibody, 2.1 mg/kg Sc209-DM4 (50 μg DM4 eq.), 6.2 mg/kgSc209-DM4 (150 μg DM4 eq), and 12.4 mg/kg Sc209-DM4 (300 μg DM4 eq).Tumor growth inhibition (TGI) was calculated at five days after the endof treatment (Day 20) using the formula (control average volume−treatedaverage volume)×100/(control average volume).

Results.

The ES2-OV64 ovarian tumor model is an engineered tumor model with highexpression of OV64 antigen. In vitro Sc209-DM4 demonstrates potencyagainst the ES2-OV64 cells, with an LD₅₀ of 2.5 nM (see above).Treatment of tumor bearing nude mice with the naked antibody did notdemonstrate statistically significant tumor shrinkage when compared tothe control group (TGI=27.5%, p>0.05). Treatment with the DM4 toxinalone at 300 μg/kg did not produce significant efficacy in this model(TGI=21.4%, p>0.05). The low dose Sc209-DM4 toxin conjugate also did notdemonstrate significant efficacy (TGI=18%, p>0.05). However, the 150 and300 μg/kg groups of the Sc209-DM4 toxin conjugates demonstrated highlysignificant growth inhibition in this model. The TGI for the Sc209-DM4at 150 and 300 μg/kg are 75% (p<0.001) and 99.4% (p<0.001),respectively. In the 300 μg/kg group there were 7 complete responsesobserved, with a duration of response of >60 days. There was no bodyweight loss in any group, in fact there was small weight gain (3-5%) inmany of the groups, suggesting that all dose levels were well tolerated.Behavior changes were not observed in any treatment group in this study.In conclusion, this study found that administration of Sc209-DM4 resultsin dose dependent efficacy against the ES2-Ov64 model on a q3d×5schedule. The conjugate was well-tolerated at all dose levels on thisschedule.

While this invention has been shown and described with references toprovided embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

What is claimed is:
 1. A method of treating cancer that expresses OV064in a subject, comprising administering to the subject an anti-OV064antibody molecule, or an antigen binding fragment thereof, wherein theanti-OV064 antibody molecule or antigen binding fragment is conjugatedto a cytotoxic moiety, in an amount sufficient to reduce or inhibit thegrowth of the subject's cancer; wherein the anti-OV064 antibody,molecule or antigen binding fragment comprises: (a) three heavy chaincomplementarity determining regions (HCDR1, HCDR2 and HCDR3) comprisingthe following amino acid sequences: HCDR1: SEQ ID NO: 19, HCDR2: SEQ IDNO: 20, and HCDR3: SEQ ID NO: 21; and (b) three light chaincomplementarity determining regions (LCDR1, LCDR2 and LCDR3) comprisingthe following amino acid sequences: LCDR1: SEQ ID NO: 28, LCDR2: SEQ IDNO: 29, and LCDR3: SEQ ID NO:
 30. 2. The method of claim 1, wherein thecancer is ovarian cancer, breast cancer, lung cancer, endometrialcancer, pancreatic cancer, or any metastases thereof.
 3. The method ofclaim 1, wherein the cytotoxic moiety comprises a radioisotope, atherapeutic agent, or a tumor activated prodrug.
 4. The method of claim3, wherein the therapeutic agent is a maytansine derivative selectedfrom DM1 or DM4.
 5. The method of claim 3, wherein the therapeutic agentis a taxane, an auristatin, a duocarmycin, a calicheamicin, or anyderivative thereof.
 6. The method of claim 3, wherein the therapeuticagent is a proteosome inhibitor or a topoisomerase inhibitor.
 7. Themethod of claim 6, wherein the proteosome inhibitor is[(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(3-mercaptoacetyl)amino]propyl]-amino]butyl]Boronicacid.
 8. The method of claim 6, wherein the topoisomerase inhibitor isN,N′-bis[2-(9-methylphenazine-1-carboxamido)ethyl]-1,2-ethanediamine. 9.The method of claim 1, wherein the antibody or antigen binding fragmentthereof is selected from the group consisting of: a human antibody or anantigen binding fragment thereof, a CDR-grafted antibody, a chimericantibody, an antigen binding fragment of a CDR-grafted antibody, and anantigen binding fragment of a chimeric antibody.
 10. The method of claim1, wherein the anti-OV064 antibody molecule further comprises human orhuman derived heavy and light chain variable region frameworks.
 11. Themethod of claim 10, wherein the heavy chain variable region comprisesthe amino acid sequence of SEQ ID NO: 10 and the light chain variableregion comprises the amino acid sequence of SEQ ID NO:
 12. 12. Themethod of claim 10, wherein the anti-OV064 antibody molecule is an IgG1antibody.